User Guide DNA Fragment Analysis by Capillary Electrophoresis (English) Manual Product Insert

USER GUIDE
DNA Fragment Analysis

by Capillary Electrophoresis
Publication Number 4474504

Revision B
LearnMore
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For Research Use Only. Not intended for use in diagnostic

procedures.
For Research Use Only. Not intended for use in diagnostic procedures.
The information in this guide is subject to change without notice.
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UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN
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TRADEMARKS
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
AmpErase, AmpliTaq, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
AFLP is a registered trademark of Keygene N.V.
Millipore is a registered trademark of Merck KGaA.
2014 Thermo Fisher Scientific Inc. All rights reserved.
Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Prerequisites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Structure of this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
CHAPTER 1
Introduction to Fragment Analysis . . . . . . . . . . . . . . . . . . 15
Fragment analysis versus sequencingwhat is the difference? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Fragment analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
What can I do with fragment analysis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Types of applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Applications described in this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
What is capillary electrophoresis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Fragment analysis workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
CHAPTER 2
Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Experimental design considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

DNA polymerase enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Derivatives of Tth DNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
22
24
24
Fluorescent labeling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Singleplexing versus multiplexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Singleplexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplexing (pooling) strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplex design software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiplexing guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26
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26
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Primer design guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Primer design criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Primer design software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors affecting Tm and primer annealing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of template secondary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selective amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preferential amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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DNA Fragment Analysis by Capillary Electrophoresis
Contents
Non-specific amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Minimizing binding to other primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Post-amplification manipulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Addition of 3' A nucleotide by Taq polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
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33
33
Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dyes and chemical forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multicomponent analysis with fluorescent dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors that affect dye signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Emission and absorption (excitation) wavelengths and relative intensities . . . . . . . . . . . . . . .
Points to consider when selecting dyes for custom primers . . . . . . . . . . . . . . . . . . . . . . . . . . .
Example: selecting dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
36
36
37
38
39
39
Dye sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Creating a custom dye set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Size standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Functions of a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Size-standard peak intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting a GeneScan size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peaks not used for sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing a size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 120 LIZ Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 500 LIZ Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 600 LIZ and GeneScan 600 LIZ v2.0 Size Standards . . . . . . . . . . . . . . . . . . . .
GeneScan 1200 LIZ Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 350 ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 400HD ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 500 ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneScan 1000 ROX Size Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
42
42
42
43
43
43
44
44
45
47
49
49
50
51
Ordering custom primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Testing the primers and optimizing conditions with test DNA panel . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Optimizing conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
CHAPTER 3
Optimizing PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Isolating, purifying, quantifying, and storing DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Isolating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purifying DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantifying DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storing prepared DNA before or after PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
55
55
56
56
56
Handling primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reconstituting and diluting primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantifying primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storing primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Contents
Using control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Purpose of control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guidelines for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CEPH 1347-02 Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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57
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Reaction volumes and plate types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Reaction volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using small amounts of template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Reagent concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
dNTP concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Magnesium ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Template concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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59
59
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60
Preventing competing side reactions: hot-start PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

When to use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Limitations and alternatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Thermal cycling parameters Veriti Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
AmpliTaq_Gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Time-release PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Touchdown PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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60
61
61
61
Thermal cycling parameters 9700 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AmpliTaq_Gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LMS2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Time Release PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Touchdown PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XL PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
62
62
62
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63
63
Optimizing thermal cycling parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Optimizing temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Avoiding contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR setup work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Avoiding contamination from the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Avoiding PCR product carryover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CHAPTER 4
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65
65
65
66
Optimizing Capillary Electrophoresis . . . . . . . . . . . . . . . . . 67
Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermo Fisher Scientific Genetic Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview of run modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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68
69
69
Contents
3500 Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Run modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating a custom dye set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
69
69
70
70
70
72

Run module and performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73
73
73
73
74

Run modules and performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
75
75
75
75
75
310 instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Run modules and performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Dye sets and matrix standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Optimizing sample loading concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Optimizing signal intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimal detection ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Balancing size-standard and sample-peak intensities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
If signal intensity is above the detection range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
If signal intensity is below the detection range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Minimizing signal intensity variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
77
77
77
78
78
78
Optimizing electrokinetic injection parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Definition of resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing injection time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing injection voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
79
79
80
Optimizing electrophoresis conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Optimizing run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing run voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing run temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
80
81
81
82
Other factors that affect electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Laboratory temperature and humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Salt concentration, ionic strength, and conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hi-Di Formamide storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polymer handling and characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
82
82
82
82
83
Understanding spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Understanding spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evaluating the calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Q Value and Condition Number ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Understanding the matrix file (310 instruments only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
84
85
86
87
87
87
Contents
CHAPTER 5 Data Analysis with GeneMapper Software and Peak

Scanner Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
How the software processes data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precise versus accurate sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Relative sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guidelines for consistent sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autoanalysis and manual analysis (GeneMapper Software only) . . . . . . . . . . . . . . . . . . . . . .
89
89
90
90
90
91
GeneMapper Software features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Peak Scanner Software Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
GeneMapper Software peak detection settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peak Amplitude Thresholds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Smoothing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Baseline Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Min. Peak Half Width . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polynomial Degree and Peak Window Size parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of varying the Polynomial Degree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of Increasing the Window Size Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
94
94
94
94
94
94
95
96
GeneMapper Software peak start and end settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

How the GeneMapper Software performs sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Size-standard definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Step 1: Size matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Step 2: Sizing curve and sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Factors that affect sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
GeneMapper Software sizing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Least Squares method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cubic Spline Interpolation method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Local Southern method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Global Southern method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
100
100
101
102
103
Evaluating data quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Examining PQVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Criteria for a good electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Examining peak definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparing data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
104
104
104
105
105
CHAPTER 6
Microsatellite Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Overview of microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advantages of using microsatellite markers (loci) in genetic studies . . . . . . . . . . . . . . . . . .
Microsatellite motifs and distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
107
108
108
109
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Contents
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Common problems with microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Identifying stutter products in microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimating the amount of stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dinucleotide repeats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evaluating data with stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Is stutter a real problem? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
113
113
114
115
117
118
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
CHAPTER 7
Single Nucleotide Polymorphism (SNP) Genotyping . . 119
Overview of SNP genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Applications (SNP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
SNaPshot Multiplex System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Applications (SNaPshot) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
CHAPTER 8
Fingerprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Amplified fragment length polymorphism (AFLP) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
126
126
126
127
127
127
128
129
130
Terminal restriction fragment length polymorphism (T-RFLP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
131
131
131
131
Contents
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
131
132
132
132
132
Bacterial Artificial Chromosome (BAC) fingerprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
132
132
133
133
134
134
135
135
136
High coverage expression profiling (HiCEP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
136
136
136
136
136
136
137
Inter-simple sequence repeat (ISSR) PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment and primer design considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
137
137
137
138
138
138
139
139
139
141
CHAPTER 9
Relative Fluorescence Quantitation (RFQ) . . . . . . . . . . 143
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Principle of the analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Experiment and primer design recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Minimizing signal intensity variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
LOH workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precise peak detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determining relative quantities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determining relative number of molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
145
145
145
146
For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Microsatellite Instability (MSI) and Replication Error (RER) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Contents
CHAPTER 10
Additional Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 149
DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
CHAPTER 11
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Troubleshooting workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
10
Checking data quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sizing Quality (SQ) PQV description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Checking samples with yellow and red SQ samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Examining the raw data for red SQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Examine the sample info, raw data, and EPT trace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
153
153
153
154
154
Running controls to isolate a problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Installation standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Agarose gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DNA template control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
156
156
156
157
157
Sample issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Salt concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
158
158
158
158
Reagent and consumable issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Laboratory water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hi-Di formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ionic buffer strength
(not applicable to 3500 Series instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
159
159
159
159
159
160
160
Instrument and ambient condition issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Capillary array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pump: large bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pump: small bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pump: polymer leaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autosampler misalignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Temperature/humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Matrix/spectral Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
160
160
160
161
161
161
161
161
Symptoms you may observe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Irregular signal intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Migration issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abnormal peak morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Irregular baseline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sizing or size quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
162
162
162
162
162
162
163
163
163
Contents
GeneMapper Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Irregular signal intensity troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Migration troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Abnormal peak morphology troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Extra peaks troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
PCR troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Irregular baseline troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Instrumentation troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Sizing or Size Quality (SQ) troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing the size-standard definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Modifying the size-standard definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
182
182
182
183
GeneMapper Software troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Preamplification gel troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Desalting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Impact of high salt concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Eliminating salt concentration as the cause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Desalting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
190
190
190
190
Evaluating 310 Genetic Analyzer multicomponent matrix quality . . . . . . . . . . . . . . . . . . . . . . . . . .

Purpose of the multicomponent matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors affecting matrix quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
When to create a new matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Virtual Filter Set C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Identifying matrix problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
191
191
191
191
191
191
Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Thermal cyclers and accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Genetic analyzers and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
GeneScan size standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Matrix standards for spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Installation standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Reagent kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Other user-supplied materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Instrument documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
GeneMapper Software documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Peak Scanner Software documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Application documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Obtain SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
11
Contents
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
12
About This Guide
IMPORTANT! Beforeusingtheproductsdescribedinthisguide,readandunderstand
theinformationintheSafetyappendixinthedocumentsprovidedwitheach
product.
Revision history
Revision
A
Date
August 2012
Description
New document.
Purpose
Thisguideisintendedforcustomerswhoplan,conduct,andtroubleshootfragment
analysisapplications.
Thisguideisforusebynoviceandexperienceduserswhoperformautomated
fragmentanalysiswithanyoftheseinstruments:
AppliedBiosystems3500or3500xLGeneticAnalyzers(3500Seriesinstruments)
AppliedBiosystems3730or3730xlDNAAnalyzers(3730Seriesinstruments)
AppliedBiosystems3130or3130xlGeneticAnalyzers(3130Seriesinstruments)
310GeneticAnalyzers(310instruments)
Prerequisites
Thisguideassumesthat:
ThermoFisherScientificgeneticanalyzersandotherinstrumentsforwhich
ThermoFisherScientificprovidesinstallationservicehavebeeninstalledbya
ThermoFisherScientifictechnicalrepresentative.
ThermoFisherScientificreagentsareused.
13
About This Guide

Structure of this guide
Structure of this guide

Chapter
Subject
Introduction
1
Introduction to Fragment Analysis
Experimental Design
Core processes in fragment analysis

3
Optimizing PCR
Optimizing Capillary Electrophoresis
Data Analysis with GeneMapper Software and Peak

Scanner Software
Types of fragment analysis

6
Microsatellite Analysis
Single Nucleotide Polymorphism (SNP) Genotyping
Fingerprinting
Relative Fluorescence Quantitation (RFQ)
10
Additional Applications
Troubleshooting
11
Troubleshooting
Reference information
Ordering Information
Documentation and Support
References
Glossary
14
Introduction to Fragment Analysis
Fragmentanalysisversussequencingwhatisthedifference? .............. 15
WhatcanIdowithfragmentanalysis? ................................... 16
Whatiscapillaryelectrophoresis?....................................... 18
Fragmentanalysisworkflow ........................................... 19
Fragment analysis versus sequencingwhat is the difference?

Fragment analysis
FragmentanalysisusingThermoFisherScientificproductsinvolves:
Labelingfragmentswithfluorescentdyes.Multipledifferentcoloredfluorescent
dyescanbedetectedinonesample.Oneofthedyecolorsisusedforalabeledsize
standardpresentineachsample.Thesizestandardisusedtoextrapolatethe
basepairsizesofthesampleproductpeaks.
Amplifyingthelabeledfragmentsusingpolymerasechainreaction(PCR)ona
thermalcycler.
Separatingthefragmentsbysizeusingcapillaryelectrophoresis.
Analyzingthedatausingsoftwaretodetermine:
Size:Theanalysissoftwareusesthesizestandardineachsampletocreatea
standardcurveforeachsample.Itthendeterminestherelativesizeofeach
dyelabeledfragmentinthesamplebycomparingfragmentswiththe
standardcurveforthatspecificsample.
Genotype:Theanalysissoftwareassignsallelecallsbasedonuserdefined
makers(loci).
Figure1 Fragment analysis fluorescently labeled fragments are separated and sized
Intensity (RFU)
Size (bp)
15
Chapter1 Introduction to Fragment Analysis

What can I do with fragment analysis?
Sequencing
SequencingisthedeterminationofthebasepairsequenceofaDNAfragmentbythe
formationofextensionproductsofvariouslengthsamplifiedthroughPCR.Formore
information,refertotheDNASequencingbyCapillaryElectrophoresis|ChemistryGuide
(Pub.no.4305080).
Figure2 Sequencing fluorescently labeled nucleotides are separated and base-called
Intensity (RFU)
Base assignment

Types of
applications
Microsatellite(STR)analysis(seeChapter6,MicrosatelliteAnalysis)
Microsatellitemarkers(loci),alsoknownasshorttandemrepeats(STRs),are
polymorphicDNAlociconsistingofarepeatednucleotidesequence.Inatypical
microsatelliteanalysis,microsatellitelociareamplifiedbyPCRusing
fluorescentlylabeledforwardprimersandunlabeledreverseprimers.ThePCR
ampliconsareseparatedbysizeusingelectrophoresis.Applicationsinclude:
Linkagemapping
Animalbreeding
Human,animal,andplanttyping
Pathogensubtyping
Geneticdiversity
Microsatelliteinstability
LossofHeterozygosity(LOH)
Intersimplesequencerepeat(ISSR)
MultilocusVariantAnalysis(MLVA)
SNPGenotyping(seeChapter7,SingleNucleotidePolymorphism(SNP)
Genotyping)
ASingleNucleotidePolymorphism(SNP)markerconsistsofasinglebasepair
thatvariesintheknownDNAsequence,therebycreatinguptofourallelesor
variationsofthemarker.Applicationsinclude:
SNaPshotMultiplexKit
16

Fingerprinting(seeChapter8,Fingerprinting)
SeveralAFLPbasedtechnologiesuserestrictionenzymelengthpolymorphism
andpolymerasechainreaction(PCR)togenerateafingerprintforagivensample,
allowingdifferentiationbetweensamplesofgenomicDNAbasedonthe
fingerprint.Applicationsinclude:
Microbialgenometyping
Animalorplantgenometyping
Creationofgeneticmapsofnewspecies
Geneticdiversityandmolecularphylogenystudies
Establishmentoflinkagegroupsamongcrosses
RelativeFluorescence(seeChapter9,RelativeFluorescenceQuantitation
(RFQ))
Relativefluorescenceapplicationscomparepeakheightorareabetweentwo
samples.Commontechniquesinclude:
QualitativeFluorescence(QF)PCR
QuantitativeMultiplexPCRofShortFluorescentFragments(QMPSF)
MultiplexLigationdependentProbeAmplification(MLPA)
Applicationsinclude:
LOHintumorsamples
CopyNumberVariation(CNV)
Aneuploidydetection
Applications
described in this
guide
Theapplicationsinthisguideareidentifiedasoneofthefollowing:
Category
Description
Thermo Fisher Scientific-supported
Thermo Fisher Scientific has tested and validated this protocol on the instrument
system specified. The technical support and field application specialists have
been trained to support this protocol.
Thermo Fisher Scientificdemonstrated
Thermo Fisher Scientific has tested this protocol but has not validated for the
instrument system specified. Certain components of the protocol workflow such
as reagent kits and other protocols for preparation of reagents may not be
available through Thermo Fisher Scientific. Supporting documentation such as
application notes may be available from Thermo Fisher Scientific and/or third
parties. Limited support is available from Thermo Fisher Scientific.
Customer-demonstrated
Thermo Fisher Scientific has not tested this protocol. However, at least one
customer or third party has reported successfully performing this protocol on the
instrument system specified. Thermo Fisher Scientific cannot guarantee
instrument and reagent performance specifications with the use of customerdemonstrated protocols. However, supporting documentation from Thermo
Fisher Scientific and/or third parties may be available and Thermo Fisher
Scientific may provide basic guidelines in connection with this protocol.
17

What is capillary electrophoresis?
What is capillary electrophoresis?

Capillaryelectrophoresis(CE)isaprocessusedtoseparateionicfragmentsbysize.In
ThermoFisherScientificCEinstrumentation,anelectrokineticinjectionisusedto
injectDNAfragmentsfromsolutionandintoeachcapillary.
Duringcapillaryelectrophoresis,theextensionproductsofthePCRreaction(andany
othernegativelychargedmoleculessuchassaltorunincorporatedprimersand
nucleotides)enterthecapillaryasaresultofelectrokineticinjection.Ahighvoltage
chargeappliedtothesampleforcesthenegativelychargedfragmentsintothe
capillaries.Theextensionproductsareseparatedbysizebasedontheirtotalcharge.
Theelectrophoreticmobilityofthesamplecanbeaffectedbytherunconditions:the
buffertype,concentration,andpH;theruntemperature;theamountofvoltage
applied;andthetypeofpolymerused.
Shortlybeforereachingthepositiveelectrode,thefluorescentlylabeledDNA
fragments,separatedbysize,moveacrossthepathofalaserbeam.Thelaserbeam
causesthedyesattachedtothefragmentstofluoresce.Thedyesignalsareseparated
byadiffractionsystem,andaCCDcameradetectsthefluorescence.
Capillary array
CCD
camera
Diffraction
system
Laser
Becauseeachdyeemitslightatadifferentwavelengthwhenexcitedbythelaser,all
colors,andthereforeloci,canbedetectedanddistinguishedinonecapillaryinjection.
Thefluorescencesignalisconvertedintodigitaldata,thenthedataisstoredinafile
formatcompatiblewithananalysissoftwareapplication.
18

Fragment analysis workflow

Phase
Technology
Thermo Fisher Scientific products used
1.
Isolate DNA
Depends on sample source

and application
DNA isolation methods depend on your starting DNA source.

Refer to guidelines for your application for information on
isolating DNA.
2.
Purify DNA
Depends on sample source

and application
Go to www.lifetechnologies.com for advice on the

appropriate product to use.
3.
Quantify DNA
Dye-labeling and
fluorometric detection
Qubit Fluorometer and Quantitation Kit, go to

www.lifetechnologies.com/qubit.
4.
PCR
amplification
Dye-labeling and
amplification of fragments
using a thermal cycler
Veriti Thermal Cycler:

96-well
384-well
GeneAmp PCR System 9700:
Dual 96-well
Dual 384-well
Auto-Lid Dual 384-well
2720 Thermal Cycler
5.
Capillary
electrophoresis
Separation of fragments
based on size using a genetic
analyzer
3500/3500xL Genetic Analyzer (3500 Series instrument)

3730/3730xl Genetic Analyzer (3730 Series instrument)
3130/3130xl Genetic Analyzer (3130 Series instrument)
310 Genetic Analyzer (310 instrument)
6.
Data analysis
Sizing and optional

genotyping
GeneMapper Software
Sizing
Peak Scanner Software (available free-of-charge on

www.lifetechnologies.com)
Use this software with data generated on 3730 Series,
3130 Series, and 310 instruments. It is not compatible
with data generated on the 3500 Series instrument, which
performs fragment sizing during data collection.
19
20

Experimental Design
Experimentaldesignconsiderations..................................... 21
DNApolymeraseenzymes ............................................. 22
Fluorescentlabelingmethods ........................................... 25
Singleplexingversusmultiplexing....................................... 26
Primerdesignguidelines............................................... 29
Dyes ................................................................ 36
Dyesets ............................................................. 41
Sizestandards ........................................................ 42
Orderingcustomprimers .............................................. 52
TestingtheprimersandoptimizingconditionswithtestDNApanel ......... 52
Experimental design considerations

Considerthefollowingquestionswhendesigningyourexperiment:
Whatsequencesandmarkers(loci)areyouinvestigating?(notapplicablefor
AFLPstudies)
Whichenzymeisappropriateforyourexperiment?(seeDNApolymerase
enzymesonpage 22)
Whatistheexpectedalleledistribution?(determinefrompublishedliteratureor
fromyourowndesignandempiricaltesting)
Whatlabelingmethodwillyouuse?(seeFluorescentlabelingmethodson
page 25)
Dofragmentsizesoverlap?(seeCompensatingforoverlappingfragmentsizes
onpage 28)
Willyouevaluateonetargetperreaction(singleplex)ormultipletargetsper
reaction(multiplex)?(seeSingleplexingversusmultiplexingonpage 26)
Whatfactorsaffectthedesignofyourprimers?(seePrimerdesignguidelines
onpage 29)
Whichdyesetsarecompatiblewithyourgeneticanalyzerandareappropriatefor
thenumberofmarkersofinterest?(seeDyesetsonpage 41andSingleplexing
versusmultiplexingonpage 26)
Whichsizestandardisappropriateforthefragmentsizerangeanddyelabelsof
yoursamples?(seeSizestandardsonpage 42)
21
Chapter2 Experimental Design

DNA polymerase enzymes

Overview
Formostapplications,AmpliTaqGoldDNAPolymeraseistheenzymeofchoice.
However,ThermoFisherScientificsuppliesanumberofPCRenzymesthathavebeen
optimizedforspecificneedsaslistedbelow.Gotowww.lifetechnologies.comfor
otheravailableenzymes.
Note: AmpFlSTR,AFLP,andSNaPShotkitsincludetheappropriateDNA
polymerasefortheapplication.
Table1 PCR enzymes supplied by Thermo Fisher Scientific
DNA polymerases
Description
AccuPrime
Taq
DNA Polymerase
System
Provides reagents for amplification of nucleic acid templates with antibody-mediated hot-start for
improved PCR specificity over other hot-start DNA polymerases. Platinum anti-Taq DNA
polymerase antibodies inhibit polymerase activity, providing an automatic hot-start, while a
thermostable accessory protein enhances specific primer-template hybridization during every
cycle of PCR. This combination improves the fidelity of Taq DNA Polymerase by two-fold and is
ideal for high-throughput screening and multiplex PCR. AccuPrime Taq DNA Polymerase
broadens primer annealing temperatures, giving you optimal performance between 55C and
65C. Applications: Multiplex PCR, TOPO TA Cloning, allele-specific amplifications.
AccuPrime Taq
DNA Polymerase
High Fidelity
Amplifies nucleic acid templates using antibody-mediated hot-start, a blend of Taq DNA
Polymerase and proofreading enzyme, and AccuPrime accessory proteins for improved PCR
fidelity, yield, and specificity over other hot-start DNA polymerases. This enzyme provides:
The highest specificity and yield for the most robust PCR amplification
9-fold higher fidelity than Taq DNA polymerase alone
Minimal optimization steps, even with non-optimized primer sets
Efficient amplification of targets over a broad size range up to 20 kb
High fidelity is achieved by a combination of Platinum anti-Taq DNA polymerase antibodies that
inhibit polymerase activity, providing an automatic hot-start, and the proofreading (3-5
exonuclease activity) enzyme Pyrococcus species GB-D. The thermostable AccuPrime accessory
proteins enhance specific primer-template hybridization during every cycle of PCR, preventing
mispriming and enhancing PCR specificity and yield.
AmpliTaq DNA
Polymerase
For general use in PCR.
AmpliTaq DNA
Polymerase, LD
Low concentrations of E. coli DNA contamination, thus is better suited for amplifying DNA of
bacterial origin.
AmpliTaq DNA Polymerase is a recombinant form of Taq DNA polymerase obtained by

expressing a modified Taq DNA polymerase gene in an E. coli host. Similar to native Taq DNA
polymerase, the enzyme lacks endonuclease and 3'-5' exonuclease (proofreading) activities, but
has a 5'-3' exonuclease activity.
AmpliTaq DNA Polymerase, LD (Low DNA), is the same enzyme as AmpliTaq DNA Polymerase;
however, the LD formulation has undergone a further purification process. The purification step
insures that false-positive PCR products will be effectively minimized when amplifying bacterial
sequences. AmpliTaq DNA Polymerase, LD is especially useful for low-copy number
amplifications.
22

DNA polymerases
AmpliTaq Gold
DNA Polymerase
Description
Use in most applications because it yields PCR fragments of high specificity.
AmpliTaq Gold DNA Polymerase is a chemically modified form of AmpliTaq DNA Polymerase.
It provides the benefits of hot-start PCR (that is, higher specific product yield, increased sensitivity,
and success with multiplex PCR) without the extra steps and modifications of experimental
conditions that make hot-start impractical for high-throughput applications. AmpliTaq Gold DNA
Polymerase is delivered in an inactive state. A pre-PCR heating step of 10 to 12 minutes at 95C,
which can be programmed into the thermal cycling profile, activates the enzyme. For low-template
copy number amplifications, step-wise activation of AmpliTaq Gold DNA Polymerase, or
time-release PCR, can be useful.
GeneAmp Gold
Fast PCR Master Mix
Allows PCR to be finished in ~40 minutes.
Platinum Multiplex
PCR Master Mix
Designed specifically for endpoint multiplex PCR. It supports easy multiplexing with minimal
optimization. Amplifies up to 20 amplicons in a single reaction. Amplifies products from
50 bp to 2.5 kb.
The performance of the Platinum Multiplex PCR Master Mix over a wide range of amplicon sizes
permits the amplification of templates from 50 bp to 2.5 kb, greatly enhancing workflow flexibility.
Coupled with its 20-plex capability and absence of primer dimers, it not only provides a
high-throughput solution but also boasts high specificity through fewer non-specific primer
binding events, and hence less reaction and primer waste.
Platinum Pfx DNA

Polymerase
Ideal for amplification of DNA fragments for high-fidelity PCR applications. High fidelity is provided
by a proprietary enzyme preparation containing recombinant DNA Polymerase from Thermococcus
species KOD with proofreading (3t exonuclease) activity. Platinum antibody technology provides
a simple, automatic hot-start method that improves PCR specificity. PCRx Enhancer Solution is
included for higher primer specificity, broader magnesium concentration, broader annealing
temperature, and improved thermostability of Platinum Pfx DNA Polymerase. The PCRx
Enhancer Solution also helps optimize PCR of problematic and/or GC-rich templates. Platinum
Pfx provides:
26 times higher fidelity than Taq DNA polymerase
Amplification of fragments up to 12 kb
Room temperature reaction assembly
Applications: Amplification of DNA from complex genomic, viral, and plasmid templates; and
RT-PCR.
Unit Definition: One unit incorporates 10 nmoles of deoxyribonucleotide into acid-precipitable
material in 30 minutes at 74C.
SuperScript III
Reverse
Transcriptase (RT)
Proprietary mutant of SuperScript II RT that is active at 50C and has a half-life of 220 minutes,
providing increased specificity with Gene-Specific Primers (GSPs) and the highest cDNA yield of
all RTs. It is ideal for RT-PCR of a specific gene or generating cDNA from total or poly (A)+ RNA
sample. Like SuperScript II, it synthesizes a complementary DNA strand from single-stranded
RNA, DNA, or an RNA:DNA hybrid. SuperScript III RT is genetically engineered by the
introduction of point mutations that increase half-life, reduce RNase activity, and increase thermal
stability. Applications: array labeling, cDNA libraries, RT-PCR, primer extension, and 3 and 5
RACE. Purified from E. coli.
23

Derivatives of Tth
DNA polymerase
ThermoFisherScientificsuppliestwomodifiedformsofThermusthermophilus(Tth)
DNApolymerase:
rTthDNAPolymeraseisobtainedbyexpressionofamodifiedformoftheTth
geneinanE.colihost.
rTthDNAPolymerase,XL(ExtraLong),providesthesamefeaturesasrTthDNA
Polymerasefortargetsequencesfrom5to40kb.Aninherent35exonuclease
activityallowsforthecorrectionofnucleotidemisincorporationsthatmight
otherwiseprematurelyterminatesynthesis.
Enzyme
characteristics
Table2 Enzyme characteristics

Characteristics
High specificity
Recommended enzyme
AccuPrime Taq DNA Polymerase
High sensitivity
AmpliTaq Gold DNA Polymerase
High fidelity
Platinum Pfx DNA Polymerase
High temperatures
AmpliTaq DNA Polymerase
Multiplex PCR
Platinum Multiplex PCR Master Mix
Amplification of low-copy number template

AmpliTaq DNA Polymerase LD (for
bacterial sequences)
High specificity at high ionic strength

Amplification of extra-long fragments

(>5 kb)
rTth DNA Polymerase, XL
Pre-PCR conversion to cDNA
SuperScript III Reverse Transcriptase
Extra cycles
High magnesium ion concentration

24

Fluorescent labeling methods
Fluorescent labeling methods

IMPORTANT! Withanylabelingtechnique,useonlyThermoFisherScientificdyes.
ThermoFisherScientificprovidesspectralcalibrationmatrixstandardsthathavebeen
optimizedforourdyesets.
Otherdyes(ormixedisomersofdyes)havevariableemissionspectraandrequirea
spectralcalibrationgeneratedforthespecificdyestocorrectforthespectraloverlap
betweenthedyes.Youareresponsibleforobtainingtheappropriatespectral
calibrationreagentsandforoptimizingcustomdyesets.
Table3 Fluorescent labeling methods
5'-end labeled primer
incorporated during the PCR primer-annealing step
Fluorescent dye-labeled dUTPs or dCTPs ([F]dNTPs

incorporated during the PCR primer-extension step
Most commonly used in microsatellite analysis
Most commonly used in SNP analysis
Higher precision: Different fluorophores have different

mobilities. DNA fragments with the same 5'-end primer
and fluorophore have comparable electrophoretic
mobility, and yield sharper fragment peaks because
5-end primer labeling yields 1:1 incorporation (that is,
one fluorophore-to-one DNA fragment).
Lower precision: Fragments labeled with [F]dNTPs tend

to produce broader peaks that often appear to be split
because variable numbers of [F]dNTPs are incorporated
during PCR in variable positions on both strands.
More consistent quantitation: Every peak in an

electropherogram is made up of multiple DNA
fragments of equal size in base pairs. When using
5'-end primer labeling, every DNA fragment contributes
a single fluorophore to the total signal of a peak, and
thus the peak area is proportional to the number of DNA
molecules.
Distinct strands: By attaching different fluorophores to
the forward and reverse primers, it is possible to
distinguish between the peaks corresponding to each
strand, and between residual double-stranded products.
Lower sensitivity: Because of 1:1 incorporation (that is,
one fluorophore-to-one DNA fragment), it yields a lower
signal than [F]dNTP-labeled fragments.
Less consistent quantitation: A variable number of

fluorophores are attached to each DNA fragment in a
population. The average number of attached
fluorophores depends upon the fragment base
composition and length and upon the ratio of [F]dNTPs
to dNTPs added to the reaction mixture. Therefore, it is
not advisable to compare peak areas between
fragments labeled with [F]dNTPs for relative
quantitation studies.
Higher sensitivity: Because most fragments contain
multiple fluorophores, a given number of [F]dNTPlabeled fragments will produce a higher signal when
compared to the same number of 5'-end labeled
fragments. The increased signal strength allows you to
use smaller reaction volumes and fewer amplification
cycles during PCR.
Low cost: You can add [F]dNTPs to any PCR. You do not
need to order or synthesize fluorescently labeled
primers before each PCR and you can use [F]dNTPs
with your existing primer sets.
5-end labeled primer
Labeled nucleotides
Post-PCR end-labeling with [F]dNTPs using Klenow is an alternative to labeling during PCR (Iwahana et al., 1995; Inazuka et al., 1996). You can
also label with [F]dNTPs using traditional techniques such as random priming or nick translation.
25

Singleplexing versus multiplexing
Thefollowingfigurecomparestheresultsobtainedusing5endlabeledprimersand
[F]dNTPs.The5endlabeledprimersgivebetterresolution,but[F]dNTPsresultin
higherpeaks.Notealsotheunincorporatedfluorescentlylabelednucleotidesinthe
[F]dNTPlabeledsample.
Figure3 Comparison of 5'-end labeled primers (top panel) and [F]dNTP-labeled primers
(bottom panel)

Singleplexing
SingleplexingisaPCRtechniqueinwhichasingletargetisamplifiedinareaction
tube.Thistechniqueusesonlyoneprimerpairineachreactionanddoesnotrequireas
muchoptimizationasmultiplexing.However,singleplexingincreasesthecostand
timeperanalysis.
Multiplexing
MultiplexingisaPCRtechniqueinwhichmultipleDNAtargetsareamplifiedinthe
samereactiontube.Multiplexingusesmultipleprimerpairsineachreaction,and
requiresoptimizationtoensureprimerpairsarecompatible.
ThermoFisherScientificfluorescentmulticolordyetechnologyallowsmultiplexing.
Allelesforoverlappinglociaredistinguishedbylabelinglocusspecificprimerswith
differentcoloreddyes.Multicomponentanalysisseparatesthedifferentfluorescent
dyecolorsintodistinctspectralcomponents.
Benefits
Potential limitations
Simplifies PCR setup
Primer-oligomer formation
Increases throughput
Loss of specificity
Decreases cost per amplification
Decreased yield of specific products

Can require significant optimization
26

Multiplexing
(pooling) strategies
Strategies
Multiplexingstrategiesinclude:
PoolingsamplesafterPCR
Note: Itisgenerallyeasiertopooltheproductsfromindividuallyamplified,
fluorescentlylabeledprimerpairsthantooptimizeamultiplexPCRcontaining
multiplefluorescentlylabeledprimerpairs.Becauseprimerefficienciesvary,it
maybenecessarytoadddifferentamountsofeachindividuallyamplifiedPCR
producttoapooltoachievesimilarpeakheights.Fluorescenceintensityfrom
eachindividualdyemayalsovary.
AmplifyingmultipleproductsinasinglePCRreaction.Optionsforpoolingina
PCRreactionareillustratedinthefollowingfigure(theorangepeaksarethe
sizestandardpeaks).
1 singleplex
reaction
1 multiplex
reaction
>1 singleplex
reactions
If you pool samples after PCR:

This strategy is simpler and more flexible, but pooling
products from multiple singleplex PCR reactions often
increase the salt concentration in the loaded samples,
which can cause unwanted downstream effects (see
Desalting on page 190).
If PCR product sizes overlap, use different color dyes so
they separate during electrophoresis (see Dyes on
page 36).
Use a combination of dyes that can be detected using
one spectral matrix (one spectral calibration).
Optimize sample concentration to optimize signal
intensity for each dye (see Optimizing signal intensity
on page 77).
If you amplify multiple PCR products:

Optimize primers:
Use different dyes to label multiplex primers of
similar lengths.
Primers cannot contain large regions of
complementarity.
Primers should have similar melting temperatures
(Tm).
Before performing the PCR, perform a preliminary
check for primer compatibility and test the pairs for
successful co-amplification.
Optimize conditions for primers in singleplex reactions
before using them in multiplex reactions to ensure the
primers are suitable for your experiment.
27

Adjusting pooling ratios

Toensuresignalbalancebetweendyesinamultiplexedsample,adjustpoolingratios
asneeded.Thefigurebelowshowstheeffectofdifferentpoolingratiosonsignal
balance.Inthisexample,apoolingratioof3:1:1yieldsbalancedsignalforthethree
dyes.
1:1:3
3:1:1
1:3:1
3:1:3
1:3:3
3:3:1
Multiplex design
software
SoftwareapplicationsareavailabletoassistwiththedesignofmultiplexPCR
(HolleleyandGeerts,2009).
Multiplexing
guidelines
Compensating for overlapping fragment sizes

Ifthesizesofdifferentfragmentsoverlap,youcandothefollowingtodifferentiate
betweenthem:
Labeloverlappingproductswithdifferentdyes.
Leavethefollowingnumberofbasepairsbetweentheknownsizeranges:
Microsatelliteapplications:15to20basepairs
SNaPshotapplications:8to10basepairs
UsedifferentprimersitestoalterthePCRproductfragmentlengths.
Loadoverlappingproductsindifferentwellsorseparatecapillaryinjections
(runs).
Enzyme choice
ThehighspecificityofAmpliTaqGoldDNAPolymerasetypicallypermits
amplifyingwithelevatedMg2+concentrationsforincreasedyield.
Primer quality
Becausereagents(suchasdNTPs)areoftenlimitingduringmultiplexPCR,usinghigh
qualityprimersisparticularlyimportant.Forexample,thedecreasedspecificity(and
thustheincreasedreagentconsumption)ofonepairofdegradedPCRprimerscan
affecttheentiremultiplexreaction.Althoughyoucancompensateforadegradedpair
ofprimerstosomeextentbyincreasingtheconcentrationoftheotherprimerpairs,the
increasedcostperreactionandthedecreasedreproducibilityovertimedonotjustify
thisshorttermsolution.
Whenbuyingormakingprimers,makesurethattheyarelengthpurifiedandthat
theyarefreeofcontaminants.
28

Primer design guidelines
Primer-pair concentrations
Typically,startoutwithequalconcentrationsforallprimerpairs.
Itisoftennecessarytoadjusttheconcentrationofprimerpairsinthemultiplex
reactionuntilthepeakheightsarerelativelyeven.Increasetheprimerpair
concentrationforfragmentsshowingweakamplification.Decreasetheprimerpair
concentrationforfragmentsshowingsignificantlygreaterthanaverageamplification.
Primer-pair compatibility
WitheithersingleormultiplexPCR,evaluateprimersforcompatibility.Avoid
excessiveregionsofcomplementarityamongtheprimers.Also,selectordesign
primerswithsimilarmeltingtemperatures(Tm).
Afteridentifyingcompatibleprimerpairs,testandevaluatepairsinsingleplex
reactionsbeforeattemptinganymultiplexreactions.Youwilloftenneedtooptimize
reactionconditionsand,occasionally,youwillneedtoredesigntheprimers.
Troubleshooting multiplex PCR

Consideramplifyingseparatelyanyprimerpairthatfailstoamplifyafterits
concentrationisincreased.
Toeliminateinterferingbackgroundpeaks,try:
Swappingprimerpairsbetweendifferentmultiplexreactions
Removingprimerpairsfromthemultiplexreaction

Primer design
criteria
Optimumlength:17to25nucleotides
OptimumTm:55to65C
UsingprimerswithsimilarTmvaluesmakesitpossibletofindthermalcycling
parametersthatareoptimalforbothprimersinaprimerpair.TheTmofareaction
isinfluencedbybasecomposition,concentrationsofMg2+andK+ionsinthe
mixture,andcosolvents.
BasedontheTm,calculatetheannealingtemperature:
Ta(C)=Tm5
The2+4Rule:Tm=[(A+T)2+(G+C)4]
Avoid:
Primerdimers
Hairpins
Secondarystructures
Secondarybindingsites
Primer design
software
ThermoFisherScientificoffersOligoPerfectDesigneravailableat
www.lifetechnologies.com.
29

Factors affecting
Tm and primer
annealing
Primerannealingisinfluencedby:
Primer/templatebasecomposition
Primer/templatebaseorder
Primerortemplatesecondarystructure
Effects of base composition

GCbondscontributemoretothestability(increasedmeltingtemperature)ofprimer/
templatebindingthandoATbonds.
Toensurestableannealingofprimerandtemplatewhileavoidingproblemswiththe
internalsecondarystructureofprimersorlongstretchesofanyonebase,select
primerswitha40%to60%G+Ccontent.
Note: DesigningprimersbasedonTmandprimerlengthtoavoidprimerdimersand
gappedduplexstructuresismoreimportantthandesigningprimersbasedonactual
percentG+Ccontent.
Effects of base order

Twoprimer/templatecomplexeswithidenticalcontentmayhavedifferentTmvalues
becausebaseorderinfluencestheoverallannealingstability.Youcandeterminethe
exacteffectofbaseorderoncomplexstabilityusingthebasepairingenergieslistedin
Table4(adaptedfromSalser,1978).Largernegativevaluesrepresentmorestable
interactionsbetweenthetemplateandprimer.
Table4 Base-pairing energies (kcal/dinucleotide pair)
3' Nucleotide
5' Nucleotide
A
1.2
2.1
2.1
1.8
2.1
4.8
3.0
2.1
2.1
4.3
4.8
2.1
1.8
2.1
2.1
1.2
Example:Considerthetwosequences:3GAC5and3CGA5.Thesequence
3GAC5containedwithinaprimerwouldcontribute 4.2kcaltothebindingenergy
( 2.1kcal[3GA5]+ 2.1kcal[3AC5]= 4.2kcal).However,iftheGandCarenext
toeachother,asin3CGA5,thecontributionincreasesto 6.4kcal
( 4.3kcal[3CG5]+ 2.1kcal[3GA5]= 6.4kcal).
Note: AlthoughaGCdinucleotideatthe3endoftheprimercanstabilizethe
templateprimerbindingcomplexwhenusingthermostableenzymessuchas
AmpliTaqDNAPolymerase,a3GCcanalsoleadtofalseprimingifyoudonot
optimizePCRconditions(TopalandFresco,1976).
Effects of primer secondary structure

StringsofGsandCscanforminternal,nonWatsonCrickbasepairs(Sarocchietal.,
1970)thatdisruptstableprimerannealing.Althoughthisanomalousbehavioris
difficulttopredict,agoodgeneralruleistoavoidrunsofmorethanthreeconsecutive
Gsinprimers.
30

However,ashortrunofGsatornearthe5endofaprimerwillnotdisruptthe
stabilityofprimertemplatecomplexesbecause5positioningdoesnotleadto
involvementindisruptivesecondarystructures(forexample,primerdimerorduplex
loops).
Similarly,selfcomplementarysequenceswithintheprimercanleadtotheformation
ofhairpinstructuresthatdisruptstableprimerbindingtotemplate.Astablehairpin
canformwithjustfourG+Cbasepairsinthestemandthreebasesintheloop
(Summeretal.,1985)(Figure4).
Figure4 Secondary structures in primers
Forward and
reverse primers
Hairpin loop
sequence
Hairpin loop
sequence
Multiple binding sites
Effects of template
secondary
structure
Primersdonotbindeffectivelytotargetsequenceswithknownsecondarystructures.
Forexample,hairpinstructuresareoftenfoundinregionsofhighG+Ccontentorin
RNAsequences.Ifyoumustdesignprimerstoaspecifictargetregionwiththe
potentialforhairpinformation,youmaytryadditionofDMSOtoyourreactionor
othercommerciallyavailablekitsfordifficulttemplateamplification.
Selective
amplification
Amplificationofthedesiredtargetsequencerequiresminimizingprimerbindingto
secondarysitesintheDNAandtootherprimers.
Note: ThisappliestotemplategenomicDNA.Theprobabilityofbindingtosecondary
sitesislowerforlowcomplexitytemplates,suchasplasmidDNA.
Ideally,thebindingoftheprimertothedesiredtemplateregion:
Isstrongestatthe5end.
Generallyrequiresahigher,morenegativevalue(tomaximizebasepairing
energy)than
9.8kcal/moleatthe3end(seeTable4onpage30).
Asageneralrule,bindingatthe3endshouldbeweakerthan 9.8kcal/mole.
Preferential
amplification
Whenallelesdifferinsizebytenormorebasepairsyoumayobservepreferential
amplificationofshorterPCRproductsoverlongerones(Walshetal.,1992).Thiswill
alsooccurwhenamplifyinglowcopynumberDNAorDNAisolatedfrom
paraffinembeddedtissues.Figure5onpage32isanexampleofpreferential
amplificationoftheD5S346marker.Inboththenormal(toppanel)andtumor(bottom
panel)samples,thepeakheightofthelarger124basepair(bp)fragmentismuch
lowerthanthatofthesmaller110bpfragment.
31

IMPORTANT! Preferentialamplificationcandecreasetheaccuracyofrelative
quantitationmeasurements.
Figure5 Example of preferential amplification of the D5S346 marker. In both the normal (top
panel) and tumor (bottom panel) samples, the peak height of the larger 124-bp fragment is
much lower than that of the smaller 110-bp fragment.
Non-specific
amplification
Polymerasesrequireonlythebindingofthenucleotidesatthe3endtobegin
elongation.Ifthe3nucleotidesbindstronglytorandomregionsofthegenome
(perhapsbecauseofa3G+C),anytemplatesequencescomplementarytothe3endare
amplified.Inthiscase,specificityislostbecausetheentireprimerdoesnotspecifically
targetthegenomicregionofinterest.
Selfcomplementarysequenceswithintheprimercanleadtotheformationofhairpin
structuresthatdecreasebindingspecificity(aswellasdisruptbindingstability).
Nucleotidesinthehairpinstructurearenotavailableforbindingofthetarget
sequence.Theavailablenucleotidescanbethoughtofasformingasmaller,and
thereforelessspecific,primer.
Conversely,ifbindingisstrongestatthe5end,thetypicalbindingeventonthe
templateDNAbeginsatthe5end.Polymerases,however,cannotbeginelongation
untilthe3endbinds.Therefore,theentireprimerisusedtodistinguishamongtarget
sequences.
Also,whenperformingacomputerassistedsearchtoevaluatebindingtosecondary
sitesinthetargetDNA,considerthepotentialforgappedduplexformation.A
gappedduplexcanformwhentheprimerandtargetarecompletelycomplementary
exceptforasinglebase(Miller,Kirchoffetal.,1987;Miller,Wlodaweretal.,1987).
Note: Bindingtosecondarysitescanalsoinvolvetheformationofstable
nonWatsonCrickbasepairs(TopalandFresco,1976).Stablebasepairingismost
likelytooccurbetweenGandT,butACandGApairscanalsobestable(Hunter,
1986).Allsoftwareprogramshavedifficultymodelingthesesortsofinteractions.
Minimizing binding
to other primers
32
Complementarysequencesbetweentwoprimers,especiallyatthe3ends,canleadto
theformationofproductartifactsarisingfromamplifiedprimerdimersand
primeroligomers.Avoidprimerswithintercomplementaryregionsbetween
membersofaprimerpairorpairs.

Post-amplification
manipulations
Addingextensionsthatarenotcomplementarytothetemplateatthe5endofthe
primercanfacilitateavarietyofusefulpostamplificationmanipulationsofthePCR
productwithoutadverselyaffectingyield.Examplesinclude5extensionsthatcontain
restrictionsites,universalprimerbindingsites,orpromotersequences.
Addition of 3' A
nucleotide by Taq
polymerase
TheAmpliTaqandAmpliTaqGoldDNAPolymerases,likemanyotherDNA
polymerases,catalyzetheadditionofasinglenucleotide(usuallyanadenosine)tothe
3endsofthetwostrandsofadoublestrandedDNAfragment.Thisnontemplate
complementaryadditionresultsinadenaturedPCRproductthatisonenucleotide
longerthanthetargetsequence.APCRproductcontainingtheextranucleotideis
referredtoastheplusAform.
Incomplete 3' A nucleotide addition

Because3Anucleotideadditionrarelygoestocompletionwithoutalongextension
stepattheendofthermalcycling(thatis,onlyafractionofthefragmentsreceivethe
extranucleotide),singlebaseladdersoftenform,creatingpeakpatternsthatanalysis
softwaremightnotinterpretcorrectly(Figure6).Theresultingallelecallscanbe
inconsistent,incorrect,ormissingentirely.
Figure6 Split peaks resulting from incomplete 3' A nucleotide addition
33

Avoiding incomplete 3' A nucleotide addition

Modify
Thermal cycling conditions
Considerations
Increasing the time spent between 60 and 72C promotes 3' A nucleotide addition.
Decreasing the time spent between 60 and 72C inhibits 3' A nucleotide addition.
To use this method effectively, determine the optimal thermal cycling conditions for
each marker in each set of reaction conditions.
Promoting 3' A nucleotide addition has proven to be more successful than removing
3' A. Residual polymerase activity at room temperature (or even at 4C) is often
sufficient to catalyze enough 3' A nucleotide addition to create genotyping problems.
Many protocols increase the final extension step to 30 to 45 minutes to promote 3' A
nucleotide addition.
Magnesium ion concentration
Increasing the magnesium ion concentration promotes 3' A nucleotide addition.

Decreasing the magnesium ion concentration inhibits 3' A nucleotide.
In general, optimizing the magnesium ion concentration is best used in conjunction with
other strategies. If you choose to maximize 3'A nucleotide addition, consider using
AmpliTaq Gold DNA Polymerase at 2.5 mM MgCI2.
Tail
the 5' end of the reverse primer
Brownstein et al. (1996) found that adding additional nucleotides (a tail) to the 5' end
of the reverse PCR primer either promoted or inhibited 3' A nucleotide addition to the
(forward) labeled strand, depending on the sequence of the added nucleotides (Figure7
on page 35).
Magnuson et al. (1996) noticed a correlation between tail sequence and the amount of
3' A nucleotide addition. In particular, they found that adding a single G to the 5' end of
the reverse PCR primer generally resulted in almost complete 3' A nucleotide addition.
Therefore, using a tail to promote 3' A nucleotide addition can consistently yield a
pattern that analysis software can identify.
Reverse-primer tailing has advantages compared to other methods because it:
Works well under diverse reaction conditions
Does not require additional experimental steps
Go to www.lifetechnologies.com for information on ordering tailed primers.
34

Figure7 Tailed primers and 3' A nucleotide addition
Labeled primer
Without tailed primers

Tailed primer
With tailed primers (plus 7 bp)
Note: Ingeneral,themostreliablestrategyistopromote3Anucleotideadditionby
modifyingthermalcyclingconditionsandMg2+concentration,and(ifnecessary)by
tailingthereverseprimer.
Enzymatic treatment
Ginotetal.(1996)usedT4DNApolymerasetoremovethe3Aoverhangsfrom
treatmentpooledPCRproducts.
Althougheffective,thismethodhasseriouslimitationsbecauseitrequires:
ApostPCRenzymatictreatmentstep
TitratingeachlotofT4DNApolymerasetodetermineoptimalenzyme
concentrationsandtreatmenttimes
IMPORTANT! ExcessT4treatmentcancausePCRproductdegradation.Insufficient
treatmentwillnotremovethe3overhangsandcanmakesomeallelesmoredifficultto
genotype.
Startoptimizationexperimentswith0.5to1unitofT4DNApolymerasein10Lof
pooledPCRproduct.Incubateat37Cfor30minutes.
35

Dyes
Dyes
IMPORTANT! WerecommendusingonlyThermoFisherScientificdyes.ThermoFisher
Scientificprovidesspectralcalibrationreagentsthathavebeenoptimizedforourdye
sets.
Otherdyes(ormixedisomersofdyes)havevariableemissionspectraandalsorequire
aspectralcalibrationgeneratedforthespecificdyesinusetocorrectforthespectral
overlapbetweenthedyes.Youareresponsibleforobtainingtheappropriatespectral
calibrationreagentsandforoptimizingcustomdyesets.
Dyes and chemical

forms
ThermoFisherScientificdyesareavailableinmultiplechemicalforms.Someformsare
suppliedcoupledtoprimersandothersyoucanusetolabelcustomprimersor
fragments.Eachformhasdistinctadvantagesanddisadvantagesdependinguponthe
intendedapplicationandyourlaboratorysetup.
YoucananalyzephosphoramiditelabeledfragmentswithNHSesterlabeled
fragments,butyoushouldnotcombine[F]dNTPlabeledfragmentswithanyother
labelingmethod.
Table5 Dye chemical forms
Chemical form
Purpose
Available dyes
Post-synthesis 5'-end labeling of oligonucleotides containing a 5'

Aminolink2
NED,
Phosphoramidite
reagents
Preparing custom, 5'-end labeled primers directly on any Thermo

Fisher Scientific DNA synthesizer
6-FAM, HEX, TET,

NED, VIC, PET
[F]dNTPs
Simple internal fluorescent labeling of multiple nucleotides during

PCR amplification
R6G, R110, ROX, TAMRA
Labeled primers in
reagent kits
Microsatellite and human identification applications
5-FAM, JOE,
6-FAM, HEX, TET,
NED, VIC, PET
Labeled size
standard
Generating the sizing curve to size unknown sample fragments
TAMRA, ROX, LIZ
NHS-esters
TAMRA, ROX
SNaPshot Kit dyes: dR110,

dR6G, dTAMRA, dROX
NED, VIC, and PET dye-labeled primers are available only in kits or through the Thermo Fisher Scientific Custom Oligo Service. Contact your
Thermo Fisher Scientific representative or visit our website for information on how to order custom-labeled oligonucleotides.
Matrix standards for spectral calibration available for the 310 instrument only.
For information about synthesizing labeled oligonucleotides, contact your Thermo Fisher Scientific representative.
5-FAM and JOE are available only as labeled primers in certain reagent kits.
Multicomponent
analysis with
fluorescent dyes
Fluorescentdyelabelingenablesyoutoanalyzemultipleindependentmarkers(loci)
inthesamecapillaryinjectionbyusingdifferentdyecolorsinadditiontosizeto
distinguishbetweenmarkers.
Duringdatacollectiononourgeneticanalyzers,thefluorescencesignalsareseparated
bydiffractiongratingaccordingtowavelengthandprojectedontoaCCDcameraina
predictablyspacedpattern.
36

Dyes
Althougheachdyeemitsitsmaximumfluorescenceatadifferentwavelength,thereis
someoverlapintheemissionspectrabetweenthedyes(Figure8).Tocorrectfor
spectraloverlap,thesoftwareappliesamulticomponentmatrix.Amulticomponent
matrixiscreatedwhenyouperformaspectralcalibrationforadyesetusingamatrix
standard(formoreinformation,seeDyesetsonpage 41andUnderstanding
spectralcalibrationonpage 84).
Figure8 Emission spectra of dyes
Dyes
Normalized Emission
6-FAM
VIC
NED PET
LIZ
100
80
60
40
20
0
500
550
600
650
700
Wavelength (nm)
Factors that affect

dye signal
Fluorescentdyeshavethefollowingcharacteristics:
Emissionspectrum:Theintensityofemittedlight(fluorescence)asafunctionof
thewavelengthoftheemittedlight.
Absorption(excitation)spectrum:Theintensityofemittedlightasafunctionof
thewavelengthoftheexcitinglight.
Absorption(excitation)efficiency:Ameasureoftheprobabilitythatadyewill
absorblightofacertainwavelength,asapercentageoftheprobabilityof
absorptionatthewavelengthofmaximumabsorption.
Quantumyield:Theprobabilitythatitsexcitedstatewillemitaphotonasit
decaysbacktothegroundstate.
Theabilityoftheinstrumenttodetectadyesignaldependsupon:
Theabsorptionefficiencyofthedyeatthewavelengthsoflightemittedbythe
laser
Thelaser/lightsource
Thequantumyieldofthedye
Thedyeconcentration
Theemissionandabsorptionwavelengthsofadyedependupon:
Thechemicalstructureofthedye
Thephysicalenvironment,including:
BufferpHandconcentration
Polymercomposition
WhethertheDNAitisattachedtoissingleordoublestranded
Althoughalteredbythephysicalenvironment,thewavelengthsofmaximumemission
andabsorptionforeachdyealwaysliewithinasmallwavelengthrange.
37

Dyes
Emission and
absorption
(excitation)
wavelengths and
relative intensities
Themaximumfluorescenceabsorptionandemissionwavelengthsarelistedbelowfor
ThermoFisherScientificNHSesters,dyephosphoramidites,and[F]dNTPbaseddyes.
(Theactualmaximumabsorptionandemissionwavelengthsmaydifferfromthelisted
valuesbecauseoftheinfluenceofthephysicalenvironmentuponthedye.)
Theintensityofemittedfluorescenceisdifferentforeachdye,andyoumustoptimize
sampleconcentrationtoaccountfordifferencesindyesignalstrength.
Examples:
6FAMdyeemitsastrongersignalthanNEDdye.Therefore,togenerate
signalsofequalintensity,youmustloadapproximatelythreetimesasmuch
NEDdyelabeledfragmentsas6FAMdyelabeledfragments.
VICdyeemitsastrongersignalandismorestablethanHEXdye.UseVIC
dyeforweakamplicons.
Table6 Dye Absorption max, emission max, and relative intensities
38
Dye
Absorption Max
Emission Max
Relative
Intensity
5-FAM
494 nm
530 nm
100 RFU
6-FAM
494 nm
522 nm
100 RFU
TET
(310 only)
521 nm
538 nm
100 RFU
VIC
538 nm
554 nm
100 RFU
JOE
528 nm
554 nm
50 RFU
HEX
535 nm
553 nm
50 RFU
LIZ
638 nm
655 nm
50 RFU
NED
546 nm
575 nm
40 RFU
TAMRA
560 nm
583 nm
25 RFU
ROX
587 nm
607 nm
25 RFU
PET
558 nm
595 nm
25 RFU
Intensity (not
to scale)

Dyes
Points to consider
when selecting
dyes for custom
primers
Whenyouordercustomprimers,youspecifythedyesforlabeling.Basedonthedyes
youspecify,youmustusetheappropriatedyesettoperformaspectralcalibration
(describedinDyesetsonpage 41).
Considerthefollowingwhenselectingdyes:
Onedyeisneededforthesizestandard(redororange).
Using5dyesprovides33%greaterthroughputthanusing4dyes.
UsethemostintensedyesforPCRproductswithlowrecoveryrate(fromlowerto
higherintensity:Blue>Green>Yellow>Red)(seeEmissionandabsorption
(excitation)wavelengthsandrelativeintensitiesonpage 38).
UselessintensedyesforPCRproductwithgoodrecoveryrate.
Selectdyeswithabsorptionmaximathatareasfarapartaspossibletoavoid
overlapandforeasiergenerationofmatrix/spectralcalibration(seeEmission
andabsorption(excitation)wavelengthsandrelativeintensitiesonpage 38).
Considertherelativedyeintensitiesandsampleconcentration(seeEmissionand
absorption(excitation)wavelengthsandrelativeintensitiesonpage 38).
Example: selecting
dyes
Thefollowingfigureshowsthemarkerrange(alleledistribution)andallele
frequenciesforthealligatormicrosatellitelocusAmi8forsamplestakenfrom
Florida/SouthGeorgiaandTexas/Louisiana.
Figure9 Allele distribution for alligator marker Ami-8 in two populations (124 to 156 bp)
Usingahypotheticalsetof10markersasanexample:
Foreachmarker,determinetheexpectedalleledistribution,eitherfrompublished
literatureorfromempiricaltesting.
Determinethedyesthatareappropriatefortherangeofeachmarkerofinterest.
39

Dyes
Forthishypotheticalsetofmarkers,youmightselect6FAM,VIC,PET,
NEDdyes,andLIZdyeforthesizestandard(seethetablebelow).Thisgroup
ofdyescorrespondstotheG5dyeset,soyouwouldalsoneedtheDS33matrix
standardforspectralcalibration(seeTable7onpage41forthematrixstandard
thatcorrespondstoeachdyeset).
Notethatdifferentdyescanbeusedforsimilarfragmentlengths,andthesame
dyecanbeusedforfragmentsofdifferentlengths.
40
Locus
Marker
Range
Dye
Marker 1
90104
6-FAM
Marker 2
112146
VIC
Marker 3
119177
PET
Marker 4
117202
NED
Marker 5
156190
6-FAM
Marker 6
221253
6-FAM
Marker 7
234282
NED
Marker 8
260342
VIC
Marker 9
311327
6-FAM
Marker10
340380
NED
Capillary electrophoresis array view

of example 10-marker panel
Dye sets
Dye sets
Dye sets and
matrix standards
Adyesetcorrespondstothegroupofdyesyouselectforlabeling(describedinthe
previoussection).Youusethematrixstandardthatcorrespondstothedyeset(shown
below)toperformaspectralcalibration.Thiscalibrationpreparestheinstrumentfor
detectionofthedyeswithwhichyourprimersarelabeled.Forinformationonspectral
calibration,seeUnderstandingspectralcalibrationonpage 84.
Table7 Dye set and matrix standard components
Dye Set
(ROX, LIZ, and TAMRA dyes are reserved for the size standard)
Dye Set
E5
G5
Matrix
standard
DS-02
DS-30
DS-31
DS-32
DS-33
DS-34
Blue
dR110
6-FAM
6-FAM
5-FAM
6-FAM
6-FAM
Green
dR6G
HEX
VIC
JOE
VIC
TET
Yellow
dTAMRA
NED
NED
NED
NED
HEX
Red
dROX
ROX
ROX
ROX
PET
TAMRA
Orange
LIZ
LIZ
Used on 310 instruments only.

Can be used for custom-labeling primers.
DS-30 versus DS-31: VIC dye emits a stronger signal and is more stable than HEX dye. Use VIC dye for
weak amplicons.
ThekitsavailablefromThermoFisherScientificusethedyesetslistedbelow.
Table8 Dye sets and matrix standards for kits and genotyping applications
Dye Set
Matrix
Standard
SNaPshot Primer Focus, SNaPshot Multiplex
E5
DS-02
Custom oligos
DS-30
Custom oligos, Plant and Microbial AFLP, Bovine and

Canine Stockmarks
DS-31
Stockmarks, AFLP
DS-32
Equine Stockmarks, custom oligos, AmpFlSTR
G5
DS-33
Application
Creating a custom
dye set
Scientificprovidesspectralcalibrationreagentsthathavebeenoptimizedforourdye
sets.
NonThermoFisherScientificdyes(ormixedisomersofdyes)havevariableemission
spectraandalsorequireaspectralcalibrationgeneratedforthespecificdyesinuseto
correctforthespectraloverlapbetweenthedyes.Youareresponsibleforobtainingthe
appropriatespectralcalibrationreagentsandforoptimizingcustomdyesetstoensure
thedyelabelsdonotaffectPCRefficiency.
41

Size standards
However,the3500Series,3730Series,and3130Seriesinstrumentsdosupportcustom
dyesets.Forinformation,seeChapter4,OptimizingCapillaryElectrophoresison
page67.
Size standards
Functions of a size
standard
Eachunknownsampleismixedwithsizestandardbeforeelectrophoresisandrun
togetherinthesamecapillarywiththesameconditions.Sizestandardsperformtwo
functions:
Allowsizingofsamplepeaks.Asizecurveisgeneratedforeachsample.Because
thesizes(inbp)ofthesizestandardpeaksareknown,thesizesofsamplepeaks
aredeterminedthrougharelativecomparisonofmigrationspeedsduring
electrophoresis.Theuniformspacingofsizestandardfragmentsensuresprecise
sizingthroughoutthesizingrange.
IMPORTANT! Becausethecalledsizeforafragmentcandifferfromitsactualsize,
comparetheallelecallsinsteadofthefragmentsize.
IMPORTANT! Usethesamesizestandard,instruments,andinstrumentconditions
forallsamplesinastudy.Usingdifferentsizestandards,instruments,or
instrumentconditionsmayshiftthesizingofDNAfragments.
Precision,orreproducibility,isthemeasureofinstrumentabilitytogeneratethe
samesizeconsistentlyforagivenfragment.
Formoreinformation,seePreciseversusaccuratesizingonpage 90,
Guidelinesforconsistentsizingonpage 90,andHowtheGeneMapper
Softwareperformssizingonpage 98.
Correctforinjectiontoinjectionvariationsthatresultindifferenceswhen
comparingthesameDNAfragmentsfromdifferentcapillaries,runs,and
instruments.
Whencomparingfragmentsizeacrossinjections,ensurethatdataisanalyzed
withthesamesizingmethodandthesamesizestandarddefinition.
Size-standard peak
intensity
42
Foroptimumperformance,thesignalintensityofthesizestandardpeaksshouldbe
lowerthanorequaltothesignalintensityofthesamplepeaks.Formoreinformation,
seeBalancingsizestandardandsamplepeakintensitiesonpage 77.

Size standards
Selecting a
GeneScan size
standard
Selectasizestandardwithatleasttwofragmentssmallerandlargerthanyour
unknownsamplefragments,andwithadyethatiscompatiblewiththedyesusedfor
labelingprimers.
LIZ Size Standard, 5-dye chemistry
Expected
marker
length
ROX Size Standard, 4-dye chemistry
GS120
LIZ
GS500
LIZ
GS600
LIZ
GS1200
LIZ
GS350
ROX
GS400HD
ROX
GS500
ROX
GS1000
ROX
(page 47)
(page 50)
(page 45)
(page 47)
(page 49)
(page 49)
(page 50)
(page 51)
120 bp
400 bp
500 bp
600 bp
1000 bp
1200 bp
SNaPshot
Used with
Multiplex Kit.
For denaturing and non-denaturing applications.
Peaks not used for

sizing
Preparing a size
standard
Somesizestandardsincludepeaksthatarenotusedforsizing.Thesepeaksare
denotedwitha*inthefollowingfigures.Thesepeakscanbeusedasanindicatorof
precisionwithinarun.
1. Vortextomixthecontentsofeachsizestandardtubethoroughly,thencentrifuge
brieflytocollecttheliquidatthebottomofthetube.
2. OptimizetheratioofsampletosizestandardandHiDiformamideusingthe
valueslistedbelowasastartingpoint.
Components
Sample
Size standard
Hi-Di
Formamide
3500 Series, 3730 Series, and

3130 Series instruments
310 instrument
0.5 L per reaction
0.5 L per reaction
0.5 L per reaction
0.5 L per reaction
9.0 L per reaction
11.0 L per reaction
Hi-Di Formamide (Part no. 4311320) is purchased separately from the size standard.
3. Createamastermixofthesizestandardandformamide.
4. Addsamplesandmastermixtotubesorwells.
5. Heatthereactionmixfor3to5minutesat95C.Immediatelychillonicefor
2 to 3 minutes,thenloadsamples.
IMPORTANT! Aftersizestandardsaremixedwithformamide,runimmediately.Signal
willdecreasesignificantlyifleftatroomtemperaturefor>1dayorat2to8C
for>5days.Platescanbestoredat20Cforupto1week.
43

Size standards
GeneScan 120
LIZ Size Standard
Range:15to120bpunderdenaturingconditions
GeneScan 120 LIZ denatured fragment lengths (nt): 9 fragments
15
35
80
20
50
110
25
62
120
Thissinglestrandedsizestandardwasdesignedtoprovideaccuratesizingofshort
DNAfragments.Therefore,itisparticularlyusefulforSNPanalysis.Allfragments
havebeenoptimizedunderawidevarietyofrunconditions.
Figure10 GeneScan 120 Size Standard run under denaturing conditions
GeneScan
500 LIZ Size
Standard
Thissizestandardisrecommendedforanalysisoftriandtetranucleotide
microsatelliteloci,whichcanoftenexceed400bpinlength.
35
139
250
400
50
150
300
450
75
160
340
490
100
200
350
500
Do not use this fragment for sizing. See Peaks not used for sizing on page 43 for information.
OnlyonestrandofthedoublestrandedDNAfragmentsinthissizestandardis
labeled.Theunlabeledstranddoesnotinterferewithpeakdetectionofthelabeled
strandwhenrununderdenaturingconditions.
44

Size standards
Figure11 GeneScan 500 LIZ Size Standard run under denaturing conditions
GeneScan 600
LIZ and
GeneScan 600
LIZ v2.0 Size
Standards
Note: TheGeneScan600LIZandGeneScan600LIZv2.0SizeStandardscontain
thesamepeaks.TheGeneScan600LIZv2.0SizeStandardcanbeusedfor
normalizationon3500Seriesinstruments.
20
120
220
314
414
514
40
140
240
320
420
520
60
160
250
340
440
540
80
180
260
360
460
560
100
200
280
380
480
580
114
214
300
400
500
600
45

Size standards
Figure12 GeneScan 600 LIZ Size Standard fragments run under denaturing conditions
Optimizing the 3130 Series instrument run module

Therunmodulesprovidedwiththeseinstrumentsmayneedtobeoptimizedforuse
withtheGeneScan600LIZSizeStandard.Add100secondstotheruntimeif
needed.
Optimizing the 310 instrument run module

Therunmodulesprovidedwiththeseinstrumentshavenotbeenoptimizedforuse
withtheGeneScan600LIZSizeStandard.Add200secondstotheruntimebefore
usingthissizestandard.
46

Size standards
GeneScan 1200
LIZ Size Standard
GeneScan 1200 LIZ Size Standard denatured fragment lengths (nt): 68 fragments
20
280
560
850
30
300
580
860
40
314
600
880
60
320
614
900
80
340
620
920
100
360
640
940
114
380
660
960
120
400
680
980
140
414
700
1000
160
420
714
1020
180
440
720
1040
200
460
740
1060
214
480
760
1080
220
500
780
1100
240
514
800
1120
250
520
820
1160
260
540
840
1200
Thehighfragmentdensity(68fragments)yieldsgreatersizingprecision,and
landmarkfragmentsalloweasypeakpatternidentificationduringdataanalysis.
ThissizestandardisidealforBACfingerprinting,TRFLP,VNTR,STR,andmany
otherDNAfragmentanalysisapplications.
Figure13 GeneScan 1200 LIZ Size Standard run under denaturing conditions
47

Size standards
Downloading 3130 instrument run modules from our website

Updatedrunmodulesforthe3130SeriesinstrumentandtheGeneScan1200LIZ
SizeStandardareavailableonourwebsite.Beforeusingthedownloadedrunmodules,
adjustasdescribedbelow.Furtheroptimizationmaybenecessary.
36-cm array with POP-4 polymer
50-cm array with POP-4 polymer
Decrease run voltage to 8000 volts
Decrease run voltage to 12,000 volts
Increase run time to 6000 seconds
Downloading 3730 instrument run modules from our website

Updatedrunmodulesforthe3730SeriesinstrumentandtheGeneScan1200LIZ
SizeStandardareavailableonourwebsite.Beforeusingthedownloadedrunmodules,
adjustasdescribedbelow.Furtheroptimizationmaybenecessary.
50-cm array
Decrease run voltage to 8000 volts
48

Size standards
GeneScan 350
ROX Size
Standard
GeneScan ROX 350 denatured fragment lengths (nt): 12 fragments
35
139
250
50
150
300
75
160
340
100
200
350
GeneScan 400HD
ROX Size
Standard
340
350
300
200
139
100
150
160
50
35
75
Figure14 GeneScan 350 Size Standard run under denaturing conditions
ThissizestandardusesROXdye.Thehighdensityofmarkerbandsinthisstandard
makesitparticularlyusefulformicrosatelliteanalysis.
GeneScan ROX 400HD denatured fragment lengths (nt): 21 fragments
50
160
260
360
60
180
280
380
90
190
290
400
100
200
300
120
220
320
150
240
340
strand.
49

Size standards
Figure15 GeneScan 400HD Size Standard run under denaturing conditions
GeneScan
500 ROX Size
Standard
Thissizestandardisrecommendedforanalysisoftriandtetranucleotide
microsatelliteloci,whichcanoftenexceed400bpinlength.
GeneScan 500 ROX denatured fragment lengths (nt): 16 fragments
35
139
250
400
50
150
300
450
75
160
340
490
100
200
350
500
50

Size standards
Figure16 GeneScan 500 ROX Size Standard run under denaturing conditions
GeneScan 1000
ROX Size
Standard
Range:
100to900bpundernondenaturingconditions
100to539bpunderdenaturingconditions(verifiedwithPOP4polymeronly)
GeneScan 1000 ROX non-denatured fragment lengths (nt): 17 fragments
47
93
292
695
51
99
317
946
55
126
439
82
136
557
85
262
692
If run under denaturing conditions (Figure17 on page 52), fragments run 18 nucleotides shorter than the
lengths listed above and some or all of the peaks appear split.
BothstrandsoftheGeneScan1000ROXSizeStandardfragmentsarelabeledand
areusedfornondenaturingapplications.
51

Ordering custom primers
Figure17 GeneScan 1000 ROX Size Standard run under denaturing conditions. Fragments
run 18 nt shorter than lengths obtained under non-denaturing conditions (see the table on the
previous page). Under denaturing conditions, sizing is not accurate above 539 nt, therefore the
and 674-nt (corresponds to the 692-nt) peak is not shown in the figure.
Note: Underdenaturingconditions,thetwostrandsofthisdoublylabeled
sizestandardmigrateatdifferentrates,appearingassplitpeaks.Toensuresizing
precisionandareliablesizestandarddefinition,youmustdefineonepeakfromeach
splitpeakpairinthesizestandarddefinition.
Ordering custom primers

Youcanobtaincustom5endlabeledprimersfromtheThermoFisherScientific
CustomOligoService.Forinformation,seeourwebsite.
Orderlabeledandunlabeledprimerpairsforthemarkersofinterest.
Testing the primers and optimizing conditions with test DNA panel
Testing
Beforeusingprimersinananalysis,testtheprimersandoptimizesamplepreparation,
PCR,andelectrophoresisconditions.
CreateapaneloftestDNAsamplestoensurethatexpectedallelesaredetectedfor
eachmarker.UseDNAsamplesthatarerepresentativeofyouroverallstudytocapture
asmuchallelicvariationaspossible.CEPHIndividual134702ControlDNAis
availablefromThermoFisherScientificandcanbeusedinyourtestDNApanel.
TestDNApanelguidelines:
Include8to16samples
Usesamplesofgoodqualitythatarewellquantified
UseequalconcentrationsofDNAsamples
Note: Ifoptimizationofsignalintensityisnecessaryforagivensample,injectthe
samplemultipletimesusingarangeofinjectionparameters.
52

Orderunlabeledprimersforthemarkersofinterestandoptimizeamplification
conditionsonyourDNAtestpanel.Youmayneedtooptimizeavarietyofparameters
includingannealingtemperature,andvariablessuchasmagnesiumconcentrationand
primerconcentrationtoensurethattheprimersworkunderuniversalconditions.
Bandsarevisualizedonagarosegelswithethidiumbromidestaining.
Optimizing
conditions
Theintensityofemittedfluorescenceisdifferentforeachdye,andyoumustoptimize
sampleconcentrationtoaccountfordifferencesindyesignalstrength.Forexample,to
generatesignalsofequalintensity,youmustloadapproximatelythreetimesasmuch
NEDdyelabeledfragmentsas6FAMdyelabeledfragments.
Formoreinformation,see:
Chapter3,OptimizingPCRonpage55
Chapter4,OptimizingCapillaryElectrophoresisonpage67
53
54

Optimizing PCR
ThischaptercontainsgeneralinformationforPCR.Forapplicationspecific
informationonPCR,seetheapplicationchapterslaterinthisguide.
Thischaptercovers:
Safetyinformation .................................................... 55
Isolating,purifying,quantifying,andstoringDNA ........................ 55
Handlingprimers ..................................................... 57
UsingcontrolDNA.................................................... 57
Reactionvolumesandplatetypes....................................... 58
Reagentconcentrations................................................ 59
Preventingcompetingsidereactions:hotstartPCR........................ 60
ThermalcyclingparametersVeritiThermalCyclers...................... 60
Thermalcyclingparameters9700ThermalCyclers ........................ 62
Optimizingthermalcyclingparameters .................................. 63
Avoidingcontamination ............................................... 64
Safety information
IMPORTANT! Foreverychemical,readtheSafetyDataSheets(SDSs)andfollowthe
handlinginstructions.Wearappropriateprotectiveeyewear,clothing,andgloves.
Note: FortheSDSsofchemicalsnotdistributedbyThermoFisherScientific,contact
thechemicalmanufacturer.
Isolating, purifying, quantifying, and storing DNA

Isolating DNA
DNAisolationmethodsdependonyourstartingDNAsource.Refertoguidelinesfor
yourapplicationforinformationonisolatingDNA.
IMPORTANT! DONOTFREEZEBLOODSAMPLESbeforeDNAisolation.Freezing
canlyseredbloodcells,andincreasetheconcentrationofPCRinhibitorsinDNA
samples.
55
Chapter3 Optimizing PCR

Isolating, purifying, quantifying, and storing DNA
Purifying DNA
Thequality,accuracy,andamplifiedlengthofaDNAfragmentcanbesignificantly
affectedbycharacteristicsofthesampleitselfandthemethodusedforpurification.
IMPORTANT! ThesuccessofAFLPanalysisisparticularlydependentuponthe
qualityofDNA.
Selectamethodbasedonthesamplesourceortissuetype,howitwasobtainedfrom
itssource,andhowitwashandledorstoredbeforepurification.Goto
www.lifetechnologies.comforthelatestinformationonDNApurification.
Quantifying DNA
ForPCRwithcustomprimers,optimizeDNAconcentrationforyourapplication.
Concentrationmayrangefrom10to100ngofpurifiedDNAperreaction.
ItisalmostalwaysnecessarytodilutePCRamplificationproductsbeforeaddingthem
tothesampletube.Typically,therequireddilutionis1:3to1:80(PCRproductto
distilled,deionizedwaterorHiDiFormamide).
BeginbyoptimizingPCRrunconditionsforyourspecificapplication.Thenruna
dilutionseriesonyourinstrumenttodeterminetheoptimaldilution.Alternatively,
run1LofPCRproductonaminigel.If,afterethidiumbromidestaining,theproduct
signalisvisiblebutnotoversaturated,trya1:10dilution.
Afterdeterminingtheoptimaldilutionratio,youcanusethesamedilutionsfor
subsequentanalysesbecausePCRyieldsshouldbefairlyconsistent.Anychangesto
thePCRconditionsortheprimerdesignmayrequiredifferentdilutions.
Note: Differentdyesemitdifferentfluorescentintensities.Therefore,PCRproduct
concentrationsmayneedoptimizationdependingonrelativefluorescenceintensity
duringelectrophoresis.SeeEmissionandabsorption(excitation)wavelengthsand
relativeintensitiesonpage38.
Storing prepared
DNA before or after
PCR
56
Storethepreparedsamplesat20Cto4Cuntilyouperformcapillary
electrophoresis.

Handling primers
Handling primers
Reconstituting and
diluting primers
Primersarecommonlyshippedinalyophilizedstate.Theunitsofalyophilizedprimer
aregivenasamass,inpicomoles.
Tocreateastockofprimersorprobe,reconstitutetheprimerorprobeinsterile
1 TE buffer(1mMTris,0.1mMEDTA,pH8.0)orsterile,nucleasefreewater.
Quantifying
primers
Storing primers
Measuretheprimerquantitywithaspectrophotometerusingaprimerspecific
absorptioncoefficient.
20Cto80Cforstocksolution(undiluted,keepconcentrationashighas
possible)
+4Cforworkingsolution,dilutedappropriately(uptoonemonth)
Using control DNA

Purpose of control
DNA
ServesasapositivecontrolfortroubleshootingPCRamplification
ControlDNAallowsyoutodistinguishbetweenproblemswiththesampleDNA
(thecontrolDNAamplifiesbutsamplesdonot)andproblemswithreagents,
thermalcyclers,orprotocols(thecontrolDNAdoesnotamplify).
Allowsyoutomonitorsizingprecision
BecausethecontrolDNAisnotusedtocalculatethesizingcurve,youcanusethe
sizesobtainedduringdifferentcapillaryinjectionstoverifythatsizingprecision
(reproducibility)iswithinacceptablelimits.
Allowsyoutocorrelatethefragmentsizesthatareobtainedindifferentrunsor
ondifferentinstruments.
Guidelines for use
AmplifyatleastonecontrolDNAsampleineveryPCRrun.
IncludeatleastoneinjectionofamplifiedcontrolDNAduringeveryseriesof
capillaryruns.Useonecontrolinjectionforeveryvariationintheelectrophoresis
parameters.
CEPH 1347-02
Control DNA
CEPHIndividual134702ControlDNAisavailableforhumanstudiesfromThermo
FisherScientific(Partno.403062).
57

Reaction volumes and plate types
Reaction volumes and plate types

Reaction volumes
ReactionvolumesforThermoFisherScientificPCRthermalcyclersare5to100 L.
Using small
amounts of
template
Althoughreactiontubesusuallydonotneedtobesterilizedorsiliconized,use
autoclavedtubeswhenamplifyingwithquantities(approximately150to500pg)of
startingDNAtemplate.
AutoclavedPCRtubesareavailablefromThermoFisherScientific(seeThermal
cyclersandaccessoriesonpage193).
Plate types
Thermal Cycler
Veriti 96-Well
Thermal Cycler
Table9 Reaction plates for each thermal cycler

Block Format
Reaction Plate Type
Networking
capability
Uses
0.1 mL or 0.2 mL
Alloy VeriFlex
Blocks
Standard 0.2 mL and

Fast 0.1 mL 96-well
formats
Yes
Veriti 384-Well
Thermal Cycler
0.02 mL aluminum
single block
384-well plate
Yes
5 to 20 L high throughput,
small sample volume.
Dual 96-Well
GeneAmp PCR
System 9700
2 aluminum
0.2 mL 96-well
blocks
96-well, 0.2 mL format
No

Dual 384-Well
GeneAmp PCR
System 9700
2 aluminum
0.02 mL 384-well
blocks
No
Auto-Lid Dual
384- Well
GeneAmp PCR
System 9700
2 aluminum
0.02 mL 384-well
blocks
384-well plate
No
small sample volume with
robotic capability.
2720 Thermal
Cycler
0.2 mL aluminum
single block
No
Ideal for basic PCR using

0.2 mL reaction tubes or
96-well reaction plates.
58
10 to 80 L medium/high
throughput.
VeriFlex Blocks provide
better than gradient PCR
optimization.

Reagent concentrations
Reagent concentrations
ThefollowingfactorscanaffectoverallyieldofspecificDNAtargetsequences:
dNTPconcentration
Magnesiumionconcentration
Primerconcentration
Templateconcentration
Enzymeconcentration
dNTP
concentration
InthestandardGeneAmpPCRprotocol,theconcentrationofeachdeoxynucleoside
triphosphate(dNTP)is200M.
Inmostcases,lowerdNTPconcentrationsdonotsignificantlyaffecttheyieldofPCR
amplificationproductandwillincreasethefidelityofthePCRamplificationproduct.
However,forefficientbaseincorporation,keepthefourdNTPconcentrationsbalanced
andabovetheestimatedKmofeachdNTP(10to15M).
SomeapplicationsmightrequirehigherdNTPconcentration(especiallywhendNTP
analoguesareused).However,excessdNTPsdecreaseenzymefidelity.
Magnesium ion
DNApolymerasesrequirefreemagnesiumioninsolutionforactivity.FormostPCR
amplifications,youcanrelateproductyieldandspecificityaswellasenzymefidelity
tothefreemagnesiumion(Mg2+)concentration:
[freeMg2+]=[totalMg2+][totaldNTP]2[EDTA]
Ingeneral,anincreaseinfreemagnesiumconcentrationleadstoanincreaseinproduct
yieldbutadecreaseinspecificityandfidelity.Toidentifythemagnesium
concentrationthatgivesthebestcompromisebetweenyieldandspecificity,inthe
presenceof800MtotaldNTPconcentration,runaMgCI2reactionseriesin50M
incrementsovertherangefrom100to400MMgCI2.
Template
concentration
TheconcentrationoftemplateinasamplecanaffectthesuccessofPCRamplification.
Toomuchtemplatepromotesnonspecificbindingofprimerstosecondarysitesor
changesthepHofthereactionmix.Toolittletemplatecanresultinpooryields,
especiallyifthetemplateisdegraded.
Evenverylowtemplateconcentrations(10copies)areoftensufficientforsuccessful
PCRamplification.
IfthestartingsampleisDNA,youcanuseupto20,000copiesofthetargettostart
optimizationexperiments.Ingeneral,thistranslatesto:
1to5ngofclonedtemplate
200pgto1ngofgenomicDNA
StartoptimizationexperimentswithlessgenomicDNAifstartingsampleislimited.
Withclean,goodqualitygenomicDNA,500to1000pgofstartingsampleistypically
sufficient.
59

Preventing competing side reactions: hot-start PCR
Enzyme
concentration
FormostPCRapplications,2.0to2.5unitsofAmpliTaqGoldDNAPolymeraseis
recommendedforeach100Lreactionvolume.
Note: Toavoidtheinaccuraciesinvolvedinpipetting0.5Lamountsofenzymeinto
eachreaction,prepareafreshmastermixofreagentsandaddtheenzyme.
Preventing competing side reactions: hot-start PCR

When to use
Considerusingthehotstarttechniquewheneveryouneedtoimprovethespecificity
andsensitivityofyourPCRamplifications.Lossofspecificityandsensitivityareoften
causedbycompetingsidereactions,whichusuallyoccurduringtheprePCRsetup
period.(Acommoncompetingsidereactioninvolvestheamplificationofnontarget
sequencesinbackgroundDNAduetomisprimingortoprimeroligomerization.)
Limitations and
alternatives
Thehotstarttechniqueiscumbersome.Ifyouhavehighthroughputneeds,switching
toAmpliTaqGoldDNAPolymerasewillgivethesamebenefitsasperformingthe
hotstarttechnique,withouttheneedforusingwaxbarriersoropeningreactiontubes.
IfyouarealreadyusingAmpliTaqGoldDNAPolymerase,performingthehotstart
techniquewillnotimprovethespecificityandsensitivityofPCRamplification.
Componentsnecessaryforamplificationmustbekeptseparatelysothatcritical
reactantsdonotmixuntilreachingatemperaturesufficientlyhightosuppressprimer
selfannealingorannealingtonontargetsequences.
Note: AlthoughmanualhotstartPCRcanincreasespecificityandyield,itis
inconvenientandyoucanencounterreproducibilityandcontaminationproblems.
Thermal cycling parameters Veriti Thermal Cyclers

AmpliTaq_Gold
60
UsethisprofileforhotstartPCRinplaceoflaborintensivemethodssuchasmanual
hotstartorwaxbeadmediatedhotstarttechniques.TheHotstarttechniquehelpsto
minimizetheformationofprimerdimersornonspecificproducts,therebyincreasing
specificityandsensitivityofPCR.ThisprofilespecifiesaprePCRheatstepfor
activationofAmpliTaqGoldDNAPolymerase.

Thermal cycling parameters Veriti Thermal Cyclers
General PCR
UsethisprofileforstandardPCR.
Time-release PCR
UsethisprofilewithAmpliTaqGoldDNAPolymerase.Thismethodminimizesthe
prePCRactivationstepandaddsaminimumof10additionalcycles,allowingfor
slowactivationoftheenzymeduringcycling.Thisprovidesasimplemethodwhere
polymeraseactivityincreasesmoreslowlyasproductaccumulates,improving
specificity.
Touchdown PCR
Usethisprofileiftheoptimalannealingtemperatureisnotknown.Thismethod
incrementallydecreasestheannealingtemperatureinearlycyclestomaximizethe
yieldofspecificproducts.
61
Thermal cycling parameters 9700 Thermal Cyclers
Thermal cycling parameters 9700 Thermal Cyclers

AmpliTaq_Gold
UsethisprofileforhotstartPCRinplaceoflaborintensivemethodssuchasmanual
hotstartorwaxbeadmediatedhotstarttechniques.Thehotstarttechniquehelpsto
minimizetheformationofprimerdimersornonspecificproducts,therebyincreasing
specificityandsensitivityofPCR.ThisprofilespecifiesaprePCRheatstepfor
activationofAmpliTaqGoldDNAPolymerase.
1 Hld
3 Tmp 35 Cycles
95.0
5:00
95.0
0:15
55.0
0:15
4.0
Method: AmpliTaq Gold
Start
F1
General PCR
2 Holds
72.0 72.0
0:30 7:00
F2
F3
Return
F4
F5
UsethisprofileforstandardPCR.
1 Hld 3 Tmp 35 Cycles 2 Holds
95.0
1.00
95.0
0:15
Start
95.0 94.0
12:00 0:15
55.0
0:15
Start
F3
72.0
0:30
3 Tmp 20 Cycles
89.0
0:15
55.0
0:15
Method: LMS2
F2
Time Release PCR
F3
F4
Return
F4
F5
72.0
0:30
3 Tmp x 10
72.0
0:30
55.0
0:15
Return
Start
F5
F1
3 Tmp 20 Cycles 2 Holds

89.0
0:15
55.0
0:15
72.0 72.0
0:30 10:00
Return
Method: LMS2
F2
F3
4.0
F4
F5
UsethisprofilewithAmpliTaqGoldDNAPolymerase.Thismethodminimizesthe
prePCRactivationstepandaddsaminimumof10additionalcycles,allowingfor
slowactivationoftheenzymeduringcycling.Thisprovidesasimplemethodwhere
polymeraseactivityincreasesmoreslowlyasproductaccumulates,improving
specificity.
1 Hld 3 Tmp 40 Cycles 2 Holds
95.0
1.00
Start
F1
62
F2
UsethisprofilewithLinkageMappingSetprimers.LinkageMappingSetprimersare
foranalysisofselectmicrosatellitelocifromtheGnthonhumanlinkagemap.
1 Hld 3 Tmp 10 Cycles
F1
4.0
Method: General PCR
F1
LMS2
55.0 72.0 72.0

0:15 0:30 7:00
95.0
0:15
55.0 72.0 72.0

0:15 0:30 7:00
4.0
Method: Time Release PCR Return

F2
F3
F4
F5

Optimizing thermal cycling parameters
Touchdown PCR
Usethisprofileiftheoptimalannealingtemperatureisnotknown.Thismethod
incrementallydecreasestheannealingtemperatureinearlycyclestomaximizethe
yieldofspecificproducts.
2 Tmp x 20
94.0
0:15
2 Tmp x 10
94.0
0:15
65.0
0:30
*
Start
F1
XL PCR
55.0
0:30
Method: Touchdown PCR

F2
F3
Return
F4
F5
Usethisprofileforamplificationof5to40kbPCRproductsusingrTthDNA
Polymerase,XLanduniquereactionconditions.Byprovidinglongertemplates,XL
PCRcomplementstechnologiesforrapid,longrangePCR.Morecompletegenescan
beamplifiedinonereactionfromknownexpressedsequences,allowingmoreintrons
tobespanned.YoucanuseXLPCRtoamplifythecontroltarget(a20.8kbproduct
fromLambdaDNA)suppliedinthekit.
1 Hld 2 Tmp X 16 2 Tmp X 12
94.0 94.0
1:00 0:15
68.0
10:00
Start
F1
94.0
0:15
2 Holds
72.0
68.0 10.00
10.00
*
Return
Method: XL PCR
F2
F3
4.0
F4
F5
Optimizing thermal cycling parameters

Optimizing
temperature
SixindependenttemperatureblocksareavailablefortheVeritiThermalCycler.Each
blockprovidesprecisecontroloverthermalcyclingparameteroptimization.For
information,refertoourwebsite.
Tofindtheoptimalthermalcyclingparameters,performaseriesofrunsvaryingthe
annealingordenaturationtemperaturesin2Cincrements.
Note: Donotvarymorethanoneparameteratatime.
Annealing temperature
change
Positive effects
Negative effects
Decreased
Increased PCR product yield
Increased amplification of
non-specific products
(background)
Increased
Increased PCR specificity
Reduced PCR yield
63

Avoiding contamination
Guidelines
Thefollowingtablesummarizestheeffectsofmodifyingtemperaturecontrol
parametersonPCRperformance.
Change in thermal cycling parameter
Increase denaturation temperatures
(up to 96C)
Effect on PCR performance

Can be necessary to allow denaturation,
especially with G+C-rich templates
Can also cause template degradation by
depurination
Decrease annealing temperatures
Can increase yield, but can reduce specificity
Increase annealing temperatures
Increases specificity, but can reduce yield
Set the denaturation, annealing, and

extension step to at least:
Allows samples to reach thermal equilibrium

at each stage
15 seconds (preferably 30 seconds) with

the GeneAmp PCR System 9700
45 seconds using thin-walled tubes with
the DNA Thermal Cycler
Use the autoextension (or AutoX) function
of a thermal cycler to allow longer
extension times in later cycles
Increases yield by allowing complete extension

of PCR product in later cycles
For most applications, an extension temperature of 72C is effective and rarely requires optimization. In the
two-temperature PCR process, the combined annealing/extension step temperature should range from
60 to 70C.
PCRprotocolsareextremelysensitivetocontaminantsintheDNA.Althoughmany
protocolsdescribesimpleorfastextractionorpurificationmethods,carefully
evaluateanychangesorimprovementsinextractionorpurificationmethods.(Also,be
surethatthephysicalandchemicalconditionofthesampleitselfareadequateforthe
intendedlabelingandassaymethods.)
PCR setup work

area
IMPORTANT! TheseitemsshouldneverleavethePCRSetupWorkArea.
Calculator
Gloves,disposable
Markerpen,permanent
Microcentrifuge
Microcentrifugetubes,1.5mL,or2.0mL,orotherappropriatecleantube(for
MasterMixpreparation)
Microcentrifugetuberack
Pipettetips,sterile,disposablehydrophobicfilterplugged
Pipettors
Tubedecapper,autoclavable
Vortex
64

Amplified DNA
work area
IMPORTANT! PlacethethermalcyclersintheAmplifiedDNAWorkArea.
Youcanusethefollowingsystems:
Veriti96WellThermalCycler
GeneAmpPCRSystem9700withtheSilver96WellBlock
GeneAmpPCRSystem9700withtheGoldplatedSilver96WellBlock
Avoiding
contamination
from the
environment
Toavoidgeneralcontamination,takethefollowingprecautionarymeasures:
Changepipettipsbetweensamples.
Usefilterpluggedpipettips.
Cleananyworkcontaminatedsurfaceusingaclothsoakedwith50%bleach.
IMPORTANT! Beforecleaningthesampleblockofathermalcycler,refertothe
instrumentuserguidefortheproperprocedure.
Closesampletubeswhennotusingthem.
AlwaysrunanoDNAnegativecontrol.
AnegativecontrolcontainsnotemplateDNA,onlyprimersandtheDNAdiluent
(usuallywaterorbuffer).
Aliquotreactionreagentstominimizethenumberoftimesyouuseastock
solution.
Avoiding PCR
product carryover
PCRproductcarryoveristhecontaminationofanunamplifiedsamplewithpreviously
amplifiedDNA.
Why carryover is a particular concern

PCRproductcarryoverisaparticularconcernbecauseamplifiedPCRproductisan
idealtemplateforsubsequentamplificationsofthatsametarget.
AsinglePCRamplificationproducesalargenumberofcopies(asmanyas1013).The
inadvertenttransferofevenatinyvolumeoraerosolofamplifiedproductcan
significantlycontaminateunamplifiedsamples.Contaminationcanresultin
falsepositivesandthedetectionandamplificationofthecontaminatingsequence
insteadofthetargetsequence.
Precautionary measures
Usepositivedisplacementpipettesorfilterpluggedpipettetips.
Physicallyseparatereactionsbeforeandafteramplification.
HandlepreandpostPCRsolutionswithseparatesetsof:
Pipettes
Pipettetips
Microcentrifugetubes
Gloves
UseAmpEraseUNGinreactionmixturestopreventthesubsequent
reamplificationofdUcontainingPCRproducts.
65

For more
information
66
ThermoFisherScientificsuppliestheGeneAmpPCRCarryoverPreventionKit
(Part no. N8080068)andAmpEraseUNG(Partno.N8080096)toensurethatPCR
productsarenotreamplifiedinsubsequentPCRamplifications.
Optimizing Capillary
Electrophoresis
Safetyinformation .................................................... 68
Overview............................................................ 68
3500Seriesinstruments ................................................ 69
310instruments....................................................... 76
Optimizingsampleloadingconcentration ................................ 76
Optimizingsignalintensity............................................. 77
Optimizingelectrokineticinjectionparameters ............................ 78
Optimizingelectrophoresisconditions ................................... 80
Otherfactorsthataffectelectrophoresis.................................. 82
Understandingspatialcalibration....................................... 84
Understandingspectralcalibration...................................... 84
67
Chapter4 Optimizing Capillary Electrophoresis

Safety information
Safety information
Forsafetyandbiohazardguidelines,refertotheSafetysectionintheuserguidefor
yourinstrument.Foreverychemical,readtheSafetyDataSheets(SDSs)andfollowthe
Overview
Note: Performcapillaryelectrophoresisunderwellcontrolledconditionswith
standardoperatingprocedures.Werecommendusingadedicatedinstrument
platformforanexperimenttominimizerandomerrorduetosizingimprecision.
Thischaptercontainsgeneralinformationforcapillaryelectrophoresis.For
applicationspecificinformationoncapillaryelectrophoresis,seetheapplication
chapterslaterinthisguide.
Thermo Fisher
Scientific Genetic
Analyzers
Table10 Thermo Fisher Scientific Genetic Analyzers (capillary electrophoresis technology)
Instrument
Number of
capillaries
3500
3500xL
24
3730
48
3730xl
96
3130
3130xl
16
310
Capillary
array
length
36 and
50 cm
Polymer
type
POP-7
POP-4
POP-6
Sample capacity
96-well plates and
8-tube strips
Data Collection Software

version
3500 Series Software v1.0
or later
96- and 384-well plates

and 8-tube strips
36 and
50 cm
POP-7
POP-6

v3.0 or v3.1 or later
22, 36, and

50 cm
POP-7
POP-4
POP-6

v3.0 or v3.1 or later
47 cm
POP-4
POP-6
Up to 48 or 96 sample
tubes
310 Data Collection

Software
3500 Series instruments: 36-cm capillary arrays and POP-4 polymer are used for HID applications only.
Formoreinformationoninstruments,seeInstrumentdocumentationonpage199.
68

Overview of run
modules
A run module contains electrophoresis parameters, such as oven temperature, detector

cell temperature, ramp rate, injection time and voltage, and run time or run voltage.
Each run module provided with the Data Collection Software has been optimized for a
specific instrument, polymer, capillary, and sample configuration (run modules for the
3500 Series instrument are embedded in the instrument protocols). Some settings, such
as injection time/voltage or run time, may need to be optimized for your instrument
and application.
Updated versions of run modules for your instrument may be available on our
web site.
Using controls
To simplify troubleshooting, run controls with every run for multicapillary

instruments or each set of runs on 310 instruments.

The 36-cm capillary arrays and POP-4 polymer are used for HID applications only.
Run modules
Table11 3500 Series instrument run modules

Configuration
23 hours Throughput
Performance
Run modules type

and
run modules name
Fragment analysis
Sizing Precision
Capillary
length
Polymer
Run
Time
3500
3500xL
Range
50 cm
POP-7
40 min
280
840
40 to
520
<0.15
<0.30
NA
50 cm
POP-6
100 min 112
336
20 to
550
<0.15
<0.30
NA
50 cm
POP-7
125 min
88
360
40 to
700
<0.15
<0.30
<0.45
36 cm
POP-4
35 min
312
936
60 to
400
<0.15
NA
NA
36 cm
POP-7
26 min
424
1272
60 to
400
<0.15
NA
NA
50 cm
POP-7
30 min
376
1104
40 to
120
<0.50
NA
NA
FragmentAnalysis50_POP7
Fragment analysis
FragmentAnalysis50_POP6
Long fragment analysis
LongFragAnalysis50_POP7
HID
HID36_POP4
HID
HID36_POP7
SNaPshot
SNaPshot50_POP7
50 bp- 401 bp- 601 bp400 bp 600 bp 1200 bp
Throughput (samples/day): The total number of samples run in 23 hours (0.5 hour for user interaction and 0.5 hours for warm-up time).
Resolution range: The range of bases over which the resolution (peak spacing interval divided by the peak width at half-max in a GeneScan
600 LIZ or GeneScan 1200 LIZ Size Standard sample sized with a third order fit) is 1. The table shows the resolution range in 90% of
samples.
Sizing precision: Standard deviation of sizes for one allele in the DS-33 install standard sized with the GeneScan 600 LIZ Size Standard v2.0
across multiple capillaries in the same run. For one injection to pass, 100% of the alleles in that injection must meet the intra-run sizing
precision specifications. The table shows the sizing precision of 100% of alleles in 90% of samples.
Not applicable because of the size of the fragments collected in the run.
69

Performance
Table12 3500 Series instrument resolution, largest fragment, and sizing

Sizing Precision of 100% of Alleles
in 90% of Samples
General
Multirun Sizing of 100% of Alleles in

90% of Samples
Resolution
Range in 90%
of Samples
Largest
Fragment
Collected in
90% of
Samples
50400 bp
401600
bp
6011,200
bp
50400 bp
401600
bp
6011,200
bp
40 to 520
600
<0.15
<0.30
NA
<1 bp
<2 bp
NA
20 to 550
600
<0.15
<0.30
NA
<1 bp
<2 bp
NA
40 to 700
1,200
<0.15
<0.30
<1 bp
<2 bp
<3 bp
60 to 400
420
<0.15
NA
NA
<1 bp
NA
NA
60 to 400
420
<0.15
NA
NA
<1 bp
NA
NA
40 to 120
120
<0.50
NA
NA
<1 bp
NA
NA
Dye sets and

matrix standards
Table18onpage86liststhedyesetsandmatrixstandardseachinstrument.
Creating a custom
dye set
Scientificprovidescalibrationreagentsthathavebeenoptimizedforourdyesets.
spectraandrequireaspectralcalibrationgeneratedforthespecificdyestocorrectfor
thespectraloverlapbetweenthedyes.Youareresponsibleforobtainingthe
1. IntheDyeSetlibrary,clickCreate.
2. Enteradyesetname.
3. SelectachemistryandtheAnyDyedyesettemplate.
4. Selectthedyecolorstouseandsetthecalibrationpeakorder:
70

a. Selectthedyecolorstouse,whichspecifiestheordernumberofthedyeused
internallybythesoftware.Notethatwhenyoudeselectadye,theorder
numberofthedyeusedinternallybythesoftwarechanges.Theexamples
belowarefora3500Seriesinstrumentwith6dyesupport,butthelogic
appliesto4and5dyes.
InExample1withalldyesselected,internalordernumberisBlue(1),
Green(2),Yellow(3),Red(4),Purple(5),Orange(6).
InExample2withthePurpledyedeselected,internalordernumberis
Blue(1),Green(2),Yellow(3),Red(4),Orange(5)theinternalorder
numberofOrangechangesto5.
InExample3withtheBlue,Yellow,andPurpledyesdeselected,internal
ordernumberisGreen(1),Red(2),Orange(3)theinternalorder
numberofGreenchangesto1,Redchangesto2,andOrange
changes to 3.
b. Specifytheorderofthepeaksinthecalibrationstandardyouareusing.Use
theinternalordernumberofthedyebasedonthedyesselected.
IMPORTANT! TheCalibrationPeakOrderfieldsdonotcorrespondtothedye
colorsdisplayedabovetheCalibrationPeakOrderfields.
InExample1onthenextpage,iftheorderofthepeaksinthe
calibrationstandardyouareusingisOrange,Red,Yellow,Blue,Green,
Purple,specifyforCalibrationPeakOrder:6(Orange),4(Red),
3 (Yellow),1(Blue),2(Green),5(Purple).
InExample2iftheorderofthepeaksinthecalibrationstandardyou
areusingisOrange,Red,Yellow,Blue,Green,specifyforCalibration
PeakOrder:5(Orange),4(Red),3(Yellow),1(Blue),2(Green).
71

InExample3iftheorderofthepeaksinthecalibrationstandardyou
areusingisOrange,Red,Green,specifyforCalibrationPeakOrder:
3 (Orange),2(Red),1(Green).ExpandtheParameterssection,then
specifyremainingsettings.
5. PerformaspectralcalibrationusingtheAnyDyedyeset.
6. Createaninstrumentprotocolthatspecifiesthecustomdyeset,thenspecifythe
instrumentprotocolinanassay.
For more
information
72
Refertothespecificationsheetforyourinstrumenttoselectacombinationofcapillary
arrayandpolymerthatprovidetherequiredresolution(seeInstrument
documentationonpage199).


Note: Becauseofthecloseproximityofcapillariesonthe96capillary3730xl
instrument,werecommendusingthe48capillary3730instrumentforfragment
analysis.
Note: Sizingprecisionmaybelowerthanexpectedforfragments<50bprunon
3730/3730xlinstrumentswithPOP7polymer.
Run module and

performance
Note: IfyouuseGeneScan1200LIZSizeStandard,downloadanewrunmodule
andoptimizeitbeforeuse.SeeDownloading3730instrumentrunmodulesfromour
websiteonpage48.
Table13 3730 Series instrument run module
3730 Analyzer
Fragment
analysis run
modules
Fragment
Analysis
Resolution
Up to 500 bp resolution with

0.15 bp sizing resolution
Runs/
day
44
3730xl Analyzer
Samples/
day
Genotypes/
day
Samples/
day
Genotypes/
day
4224
84,508
2112
42,254
20 genotypes/sample.
Dye sets and

matrix standards
Creating a custom
dye set
Thesoftwareassumesthefollowingdyecolororder:blue,green,yellow,red,orange.
1. InthenavigationpaneoftheDataCollectionSoftware,click
GA Instruments > ga3730 or ga3130> ProtocolManager.
2. IntheInstrumentProtocolspane,clickNew.TheProtocolEditoropens.
3. IntheProtocolEditor,createaspectralprotocolfortheAny4DyeorAny5Dyedye
set,specifyingtheappropriateprotocolparameters.
4. CreateaSpectralPlateRecordusingthenewlycreatedSpectralInstrument
Protocol.
5. Performaspectralcalibration.
73

6. Setthecustomdyesetcalibrationastheactivespectralcalibration:
a. InthetreepaneoftheDataCollectionsoftware,click
GA Instruments > ga3130xl orga3130 > instrument
name >
SpectralViewer.
b. IntheDyeSetdropdownlist,selectthecustomdyeset.
c. IntheListofCalibrationsforDyeSetdropdownlist,selectthespectral
calibrationyouwanttouse.Thespectralprofileandrawdataisdisplayed.
d. ClickSet.
e. Createaninstrumentprotocolthatspecifiesthecustomdyeset.
For more
information
74


Run modules and
performance
Note: IfyouuseGeneScan1200LIZSizeStandard,downloadanewrunmodule
andoptimizeitbeforeuse.SeeDownloading3130instrumentrunmodulesfromour
websiteonpage48.
Table14 3130 Series instrument run modules and resolution
24-hr throughput
Fragment analysis run

modules
Array
length
Polymer
Fragment Analysis 22_POP4
22 cm
SNP22_POP4
Performance
Run
time
3130
Analyzer
GT
3130xl
Analyzer
GT
Resolution
POP-4
20 min
5,760
23,040
400 bp
0.50
22 cm
POP-4
15 min
3840
15,360
120 bp
0.50
36 cm
POP-7
35 min
3280
13,120
500 bp
0.15
36 cm
POP-4
45 min
2560
10,240
500 bp
0.15
HID Fragment Analysis

36_POP4
36 cm
POP-4
45 min
2560
10,240
500 bp
0.15
SNP36_POP4
36 cm
POP-4
30 min
3840
15,360
120 bp
0.15
50 cm
POP-7
50 min
2240
8,960
500 bp
0.15
50 cm
POP-4
65 min
1760
7,040
500 bp
0.15
50 cm
POP-6
90 min
1280
5,120
500 bp
0.15

SD
20 genotypes/injection.
Standard deviation: 1 base pair (bp) resolution at 99.99% accuracy.
10 genotypes/injection.
Dye sets and

matrix standards
Creating a custom
dye set
Theprocedureforcreatingcustomdyesetsisthesameastheprocedureforthe3730
Seriesinstruments.Creatingacustomdyesetonpage73.
For more
information
75

310 instruments
310 instruments
Run modules and
performance
Note: IfyouuseGeneScan600LIZSizeStandard,optimizetherunmodulebefore
use.SeeOptimizingthe310instrumentrunmoduleonpage46.
Table15 310 instrument run module
Fragment
analysis run
modules
GS STR POP4
Resolution
Samples/day
Genotypes/day
1 base detection up to 250 bases with 0.15 SD
4-dye
>57
720 genotypes
2 base detection 250 to 350 bases with 0.3 SD
5-dye
>57
960 genotypes
15 genotypes/run.
20 genotypes/run.
Dye sets and

matrix standards
Optimizing sample loading concentration

Note: Sampleoverloadingcanclogcapillaries.
OptimizetheratioofsampletosizestandardandHiDiformamideusingthe
valueslistedbelowasastartingpoint.
Components
Sample
Size standard
Hi-Di
Formamide
3500 Series, 3730 Series, and

310 instrument
0.5 L per reaction
0.5 L per reaction
0.5 L per reaction
0.5 L per reaction
9.0 L per reaction
11.0 L per reaction
Hi-Di Formamide (Part no. 4311320) is purchased separately from the size standard.
Ifyouanticipateanextremelyhighsampleconcentration,rundilutionsofthe
sample.Ifthesignalistoostrong,youcanfurtherdilutethesampleoryoucan
decreasethesampleinjectiontimeand/orinjectionvoltage.
Ifthesignalistooweak,firsttryincreasingthesignalbyincreasingthesample
injectiontimeorvoltage.
Tooptimizethesignalintensityforagivensample,injectthesamesample
multipletimesusingarangeofinjectionparameters.
Ifthesignalintensityisstilltooweakortheresolutionispoor,concentratethe
sample.
76

Optimizing signal intensity
Ifthesignalintensityistoolowafterconcentration,seeDesaltingonpage190.
Differentdyesemitdifferentfluorescenceintensities.Therefore,concentrationsof
PCRproductsmayhavetobeincreasedordecreaseddependingonrelative
fluorescenceintensityduringelectrophoresis.(SeeEmissionandabsorption
(excitation)wavelengthsandrelativeintensitiesonpage38).
Optimizing signal intensity

Optimal detection
ranges
ThermoFisherScientificgeneticanalyzerscanconvertalimitedrangeoffluorescence
signalintodigitalvalues.Foroptimalresults,ensurethesignalintensitiesarewithin
therangeslistedbelow.
Table16 Signal intensity ranges and fluorescence saturation
Balancing
size-standard and
sample-peak
intensities
Instrument
Recommended signal
intensity range
Fluorescence saturation
3500 Series
17510,000 RFU
30,000 RFU
3730 Series
15010,000 RFU
30,000 RFU
3130 Series
1504,000 RFU
8000 RFU
310
1504000 RFU
8000 RFU
Theintensityofsizestandardpeaksshouldbe30to100%oftheintensityofsample
peaks.Dilutesamplesbeforepreparingcapillaryelectrophoresisreactionstobalance
thesignalintensities.Intheexamplebelow,theundilutedsampleyieldsthecorrect
sizestandardtopeakintensityratio.
Undiluted
Size-standard
peaks should be
30 to 100% of the
intensity of sample
peaks
Diluted 1:4
Diluted 1:6
77

Optimizing electrokinetic injection parameters
If signal intensity is
above the detection
range
Whenthesignalintensityofapeakistoohigh,theinstrumentcannotmeasurethetrue
valueofthesignalandconsequentlycannotcompensateforthespectraloverlap
betweenthedyes.Asaresult,artifactpeakscalledbleedthroughorpulluppeaks
canappearbeneaththesamplepeaks.Thesepulluppeakscancorruptboth
automatedsizing(becauseextrapeaksarepresentinthesizestandarddyecolor)and
theanalysisofsamples(becausethesizestandardispresentineachsample).
Ifsignalintensityishigh,youcan:
DilutethetemplatebeforePCR
Dilutetheamplifiedsamplebeforeaddingtoformamide
Decreasethesampleinjectiontimeand/orinjectionvoltage
If signal intensity is
below the detection
range
Whensignalistoolow,thesignaltonoiseratioisalsolowandmakesitdifficultto
discriminatebetweensamplepeaksandcommonbackgroundfluctuations.
Ifsignalintensityislow,youcan:
Increasethesampleinjectiontimeorinjectionvoltage
IncreasethevolumeoftemplateaddedtothePCRreaction
Minimizing signal
intensity variation
Tominimizesignalintensityvariations,considertheionicstrengthofsamplesand
consumables.TheamountofDNAinjectedisinverselyproportionaltotheionic
strengthofthesolution.Notethefollowing:
Sampleshighinsaltresultinpoorinjections.
PCRreactionsvaryinefficiency,thereforesomereactionsmayresultinhigher
ionicconcentrationpostamplification.
Conductivityofthesolventusedforinjectionaffectsthesampleinjectionandcan
causevariationinpeakheight.
Therecommendedinjectionsolvent,HiDiFormamide,ishighlydeionized
formamide,formulatedwithastabilizer.StorageofHiDiFormamideis
importantinmaintainingthequalityandconductivityofthesolvent.SeeHi
DiFormamidestorageonpage82.
Formoreinformation,seeIrregularsignalintensitytroubleshootingonpage164.

Electrokineticinjectionparametersaffectdataquality,runtorunprecisioninsizing,
andreproducibilityintheamountofsampleloaded.Optimizeparameterstoinject
sufficientDNAtoyieldpeaksofadequateheight(thatis,datawithagoodsignalto
noiseratio)whilemaintainingtheresolutionandprecisionrequiredbythe
application.
TheDataCollectionSoftwareincludesrunmoduleswithpresetvaluesforinjection
timesandvoltagesthathavebeenoptimizedforspecificinstrument/polymer/capillary
lengthconfigurations.Thesevaluesareadequateformanyapplications.However,
considermodifyingtheinjectionparametersiftherunmodulesyieldsignalthatistoo
strongortooweakoriftheresolutionispoor.(Themaximumrecommendedinjection
timeis30secondsandthemaximumpossibleinjectionvoltageis15kV.)
78

Definition of
resolution
Theresolution,Rs,oftwopeaksinanelectropherogramisdefinedas:
whereP1andP2arethepeakpositionsmeasuredbelowthepeakapexandW1andW2
arethepeakwidthsmeasuredathalfpeakmaximum.
AnRsvalueof1correspondstofragmentsthatcanbediscriminatedbyonenucleotide.
Optimizing
injection time
Injectiontimeaffectssignalintensityandresolution.
Note: Saltconcentrationcanalsoaffectsignalintensityandresolution.Ifadjusting
injectiontimeandvoltagedoesnotprovideadequatesignalstrength,youmayneedto
concentratethesampleordesaltthesample(Desaltingonpage190).
Attribute
Effect of injection time
Signal
intensity
Signal intensity (as measured both by peak height and by peak area) typically increases linearly with
increasing injection time. However, an n-fold increase in injection time does not result in an n-fold
increase in peak height. In the examples below, no improvement is seen after 10 seconds for the larger
fragment. The signal decreases dramatically after 40 seconds for the smaller fragment. As the injection
time increases, the resolution decreases, leading to increasing peak widths and decreasing peak
heights
Resolution
Increasing the injection time decreases the resolution. As shown below, the negative effect on resolution
is more pronounced for larger fragments.
The decrease in resolution results from an increase in peak width (as opposed to a decrease in peak
separation).
79

Optimizing electrophoresis conditions
Optimizing
injection voltage
Injectionvoltageaffectssignalstrength.However,lowervoltages,whichproduce
lowercurrents,areoftenpreferablebecauseinjectiontimingismoreaccurate.Accurate
timingensuresreproducibilityinsampleloading.
Note: Saltconcentrationcanalsoaffectsignalintensityandresolution.Ifadjusting
injectiontimeandvoltagedoesnotprovideadequatesignalstrength,youmayneedto
concentratethesampleordesaltthesample(Desaltingonpage190).
Attribute
Effect of injection voltage
Signal
intensity
Peak height and peak area increase linearly with increasing injection voltage. The figures below show
the effect of increasing the injection voltage from 53 V/cm to 319 V/cm on peak height and peak area,
respectively, for two different-sized fragments.
Resolution
Injection voltage has little effect on peak resolution.

Optimizingelectrophoresisconditions(runtime,runvoltage,runtemperature)can
greatlyimprovedataquality,runtorunprecision,and/orthroughput.Optimize
settingsappropriatefor:
Rangeoffragmentlengths
Requireddegreeofresolution
Typeofgeneticanalysisyouwillbeperforming(forexample,denaturingor
nondenaturingconditions)
Thesettingsintherunmodulesprovidedaresettoensurethefollowing:
Detectionofallfragmentsinthetypicalsizerangefortheapplication
Acceptableruntimes
Acceptableresolution
80

Optimizing
run time
Performtrialrunstodeterminetheminimumacceptableruntimeforagivenrun
voltage.Toensurethatyoucollectsufficientdatatoperformanalysis,setthe
electrophoresisruntimeapproximately10%higherthanthemigrationtimeofthe
largestfragmentofinterest.
Thelargestfragmentofinterestisoftenasizestandardpeakthatisneededforsizing
thelargestsamplefragmentsofinterest.Thesetofsizestandardpeaksthat
GeneMapperSoftwareusestogeneratethesizingcurvecanvarywiththesizecalling
method.Ingeneral,besuretoincludethetwosizestandardpeaksimmediately
smallerthanthesmallestfragmentandthetwosizestandardpeaksimmediately
largerthanthelargestsamplefragmentofinterest,ormodifythesizestandard
definitiontoeliminatethepeaksthatarenotpresent.
Note: Forfasterruntimes,youcanalsoincreasetherunvoltage.However,ahigher
runvoltagecandecreasetheresolution.
Optimizing
run voltage
Runvoltagecanaffectmigrationratesandresolutionbecauseitaffectsthespeedat
whichsamplesmigratethroughthecapillary.Iftheymigratetooquickly,thesamples
donotoptimallyseparate.
Attribute
Effect of run voltage
Migration
rates
Higher run voltages yield faster run times but can affect the resolution.
Resolution
In general, resolution is better at lower run voltages.
81

Other factors that affect electrophoresis
Optimizing run
temperature
Performnondenaturingapplicationsatlowertemperatures(27to42C).
ProtocolsfordenaturingapplicationsusePOP4orPOP7polymerwithoptimized
runtemperatures.Alteringtheruntemperaturecanaffectmigrationratesand
resolution.

Laboratory
temperature and
humidity
Maintainthelaboratorytemperaturebetween15to30C.Aftertheinstrumentissetup
andinoperation,thelaboratorytemperatureshouldnotfluctuatemorethan 2C.The
instrumentcantolerateupto80%noncondensingrelativehumidity.Avoidplacingit
nearheatersorcoolingducts.
Salt concentration,
ionic strength, and
conductivity
SaltanionscompetewithnegativelychargedDNAforentryintothecapillaryduring
electrokineticinjection.Asthesaltconcentrationofasampleincreases,lessDNAwill
enterthecapillary,decreasingthefluorescencesignal.Excesssaltcanalsoprecipitate
theDNAinthesampletubeinthepresenceofformamide.
TheamountofDNAinjectedisinverselyproportionaltotheionicstrengthofthe
solution(Butleret.al.)
Hi-Di Formamide
storage
CAUTION! MixingHiDiFormamidewithwatergeneratesformicacid.
ProperhandlingandstorageofHiDiFormamideiscritical.Forqualityresults:
Aliquotthecontentsfromtheoriginalbottleintoonetimeuse,1.5mLorsmaller
tubes.
Minimizeexposuretoairandfreeze/thawcycles.
IMPORTANT! Donotfreeze/thawmorethantwotimes.Excessivefreeze/thaw
cyclesorstorageat2to8Cformorethan1weekcauseshydrolysisintoformic
acidandformate.Formateionsmigratepreferentiallyintothecapillaryduring
electrokineticinjectioncausingalossofsignalintensity.
EnsurethatyoudonotcontaminateHiDiFormamidewhensettingup
samples.
82

Storeforupto3monthsat15to25C.
Storeforupto1weekat2to8C.
Thefigurebelowillustratesthevariationinconductanceindifferentquality
formamidesolutions.
Figure18 Effect of formamide quality on conductance
ImproperlystoredHiDiFormamidecancauseavarietyofelectrophoresisproblems.
Forinformation,seeHiDiformamideonpage159.
Polymer handling
and characteristics
IMPORTANT! Donotleavepolymerontheinstrumentmorethansevendays.Polymer
leftontheinstrumentformorethansevendayscausesalossofresolution.Avoid
actionsthatintroducebubblesorparticlesintothepolymer.Dustinthepolymercan
causedataspikes.
Tominimizebubblesandparticlesinpolymer:
Closethepolymercapduringstoragetominimizeexposureofthepolymertoair.
Cleanthepolymerdeliverysystemwithdeionizedwater.
Discardcapillariesthatareexposedtodustoraredriedout.
Changethebufferandwateranddiscardthewastedailyorbeforeeachsetof
runs.
Table17 Polymer characteristics
Instrument
Polymer characteristics
POP-4 Polymer
Less viscous, fast runs
POP-6 Polymer
More viscous, slow runs
POP-7 Polymer
(not for use on 310 instruments)
Less viscous, fast runs
83

Understanding spatial calibration
Understanding spatial calibration

AspatialcalibrationmapseachcapillarylocationtotheCCDcamera,whichdetects
thesignalforeachcapillary.
Capillaries
CCD camera positions
Good spatial calibration
Bad spatial calibration
Spatialcalibrationmaximizesdataqualityandaccuracy.
Refertotheinstrumentuserguideforinstructionsonperformingaspatialcalibration.
SeeInstrumentdocumentationonpage199fordocumentpartnumbers.
Performaspatialcalibrationwhenyou:
Installorreplaceacapillaryarray
Temporarilyremovethecapillaryarrayfromthedetectionblock
Movetheinstrument
Openthedetectionblock
Understanding spectral calibration

IMPORTANT! Alwaysvisuallyexaminethedatageneratedbyaspectralcalibration.
Acceptingacalibrationwithpoordatawillyieldinaccurateresultswhenyourun
samples.
Forinformationoncreatingacustomdyesetforspectralcalibration,seethe
instrumentsectionsearlierinthischapter.
84

Spectral
calibration
Aspectralcalibration(ormatrixon310instruments)allowsthesoftwaretodistinguish
betweendyesbysubtractingoutthespectraloverlapbetweenthedifferentdyes(for
moreinformation,seeMulticomponentanalysiswithfluorescentdyesonpage36).
Before spectral calibration
After spectral calibration
SpectralcalibrationmatrixstandardsareavailablefromThermoFisherScientificin
premixedformforallThermoFisherScientificinstrumentsanddyesets(seeTable18
onpage86).
Thevaluesinamatrixgeneratedbyaspectralcalibrationareuniqueforeach
instrument,foreachdyeset,andforeachspecificsetofrunconditions.TheData
CollectionSoftwareappliesthevaluesinthematrixtothesampledatatoperform
multicomponentanalysis:theseparationofthedyefluorescenceintherawdatafrom
theinstrumenttothedatastoredinthesamplefiles.
Note: For310instruments,youmustmanuallycreateandapplythematrixfileinthe
GeneMapperSoftware.
Refertotheinstrumentuserguideforyourinstrumentforinstructionsonperforming
aspectralcalibrationorcreatinga310matrix.
When to perform
Performaspectralcalibrationrun:
Whenyouuseanewdyesetontheinstrument
Whenyouchangethecapillaryarrayorpolymer
Foreachcombinationofcapillaryarraylengthandeachdyesetthatyouuse
AfterthelaserorCCDcamerahasbeenrealigned/replacedbyaserviceengineer
Ifyouobserveadecreaseinthequalityofraworanalyzeddata(forexample,
pullupand/orpulldownpeakswithadistinctpattern)
85

Dye set and matrix standards for spectral calibration

Thetablebelowliststheappropriatedyesetandmatrixcalibrationstandard
combinationsforeachinstrument.Forthedyesetsforapplications,seeTable8onpage
41.
Table18 Matrix standards (see page 197 for part numbers)
Matrix
standard
Dye set
3500
3730/
3730xl
3130/
3130xl
310
DS-33
G5
Yes
Yes
Yes
Yes
DS-02
E5
Yes
Yes
Yes
Yes
DS-32
Yes
Yes
Yes
Yes
DS-30
Yes
Yes
Yes
Yes
DS-31
Yes
Yes
Yes
Yes
DS-34
No
No
No
Yes
AnyDye
Custom dye
set you
create
Yes
Yes
Yes
No
Because of the close proximity of capillaries on the 96-capillary 3730xl instrument, we recommend using the
48-capillary 3730 instrument for fragment analysis. For best results, use the G5 dye set with reduced
cross-talk (RCT) configuration.
Evaluating the
calibration results
Usethefollowingcriteriatoevaluatethedata:
QValueorQualityValueMeasurestheconsistencybetweenthefinalmatrix
andthedatafromwhichitwascomputed.AQvalueof1.0indicateshigh
consistencyandthatnopulluporpulldownpeaksweredetected.
ConditionNumberRepresentstheamountofoverlapbetweenthedyepeaksin
theemissionspectraofthedyesinadyeset.AConditionNumberof1.0(lowest
possiblevalue)indicatesthereisnooverlapinadyeset.Theconditionnumber
increaseswithincreasingpeakoverlap.
SpectralprofileShowstheemissionspectraofthedyes.
RawdataTheemissionimageshowsdistinctfluorescenceemissionmaxima,
oneforeachdye(Figure19).
Figure19 Example output from a spectral calibration using a matrix standard
86

Q Value and
Condition Number
ranges
Troubleshooting
spectral calibration
Matrix standard
Dye set
Quality value
Maximum
Condition Number
User-provided
AnyDye
0.8 (default)
DS-30
0.95
DS-33
G5
0.95
13.5
DS-32
0.95
8.5
DS-31
0.95
DS-02
E5
0.95
Apoororincorrectmatrixresultsintoomuchortoolittlesubtractionofdyespectral
overlapduringdataanalysis.Eachcausesarecognizableelectropherogramanomaly:
Bleedthroughpeaks,alsocalledpulluppeaks(causedbytoolittlesubtraction)
Elevatedinterpeakbaseline(causedbytoomuchsubtraction)
Ifthespectralcalibrationfails,orifthequalityofapassingcalibrationisnot
acceptable,tryoneormoreofthefollowing:
Ensurethatyouusedfresh,properlypreparedandvortexedmatrixstandard.
Old,improperlyprepared,orinsufficientlyvortexedmatrixstandardcancause
lowsignalintensity.
Checkinstrumentstatusforanyrunerrors.
Verifythecorrectrunmodulewasused.Correctasneededandrepeattherun.
Checkthefreshnessandpreparationofreagents.
Checkforpossiblecontaminationofmatrixstandards.
Makesurethattherearenobubblesinthesamplewells.
Verifythatallpeaksweredetected.
Aslowrunningsystemcanpartiallyorcompletelycutoffthebluepeak.Increase
theruntime(instrumentsotherthanthe3500series)orchangereagentsifneeded
andrepeattherun.
Fortroubleshootingthespectralcalibration,refertotheinstrumentuserguideforyour
instrument.
Understanding the
matrix file (310
instruments only)
Purpose of a matrix
Themostintensefluorescenceemittedbyafluorescentlylabeleddyefallswithina
smallwavelengthdetectionrange.However,somefluorescenceemissioninthe
detectionrangesoftheotherdyeswillalwaysoccur.Youcreateamatrixthat
compensatesforoverlapbysubtractingout,inthedetectionrangeofeachdye,the
portionofthesignalduetofluorescencefromotherdyes.
Runeachrelevantdyematrixstandardseparatelytodeterminetheproportional
amountoffluorescencethatisemittedinalldyedetectionregions.Alwayscreatea
newmatrixifrunconditionschange(suchasthepHorpolymertypeand
concentration).
87

Virtual Filter Set C

VirtualFilterSetC/dyesetDS34isusedon310instruments.Theemissionmaximum
of6FAMdye,therecommendedbluedyefortheVirtualFilterSetC,isverycloseto
thelaserwavelengthof514.5nm.Thus,thewindowforcollectedbluelightintensity
dataisoffsettolongerwavelengthsanddoesnotcontaintheemissionmaximumof6
FAMdye.Theemissionmaximumof6FAMdyeisalsoveryclosetothedetection
regionforthegreenTETdye.MatrixfilesmadeforVirtualFilterSetCareespecially
susceptibletominorchangesinrunconditions.IfyouareusingVirtualFilterSetC,
watchforevidenceofmatrixproblemsandcreateanewmatrixassoonasproblems
appear.
Factors affecting matrix quality

Agingreagents
Buffertypeandconcentration
Polymertype
Denaturingornondenaturingconditions
Runtemperature
88
Data Analysis with GeneMapper

Software and Peak Scanner
Software
Overview............................................................ 89
GeneMapperSoftwarefeatures ........................................ 91
PeakScannerSoftwareFeatures ....................................... 92
Workflow ............................................................ 93
GeneMapperSoftwarepeakdetectionsettings........................... 94
GeneMapperSoftwarepeakstartandendsettings....................... 97
HowtheGeneMapperSoftwareperformssizing ......................... 98
GeneMapperSoftwaresizingmethods ................................ 100
Evaluatingdataquality............................................... 104
Overview
Thischaptercontainsgeneralinformationfordataevaluation.Forapplicationspecific
informationondataevaluation,seetheapplicationchapterslaterinthisguide.
GeneMapperSoftwareanalyzesthedatacollectedonThermoFisherScientificgenetic
analyzerstosizeandgenotypeDNAfragments.YoucanalsousetheGeneMapper
Softwaredatatoperformrelativequantitation(formoreinformation,seeChapter9,
RelativeFluorescenceQuantitation(RFQ)onpage 143).
PeakScannerSoftware,canbeusedforpreliminarysizing.
How the software

processes data
BothGeneMapperSoftwareandPeakScannerSoftwareperformanalysison
original.fsafilesgeneratedbytheDataCollectionSoftware.
.fsa files
Baselining
Peak detection
Size matching/Size curve
Genotyping
Quality Value determination
89
Chapter5 Data Analysis with GeneMapper Software and Peak Scanner Software
Overview
Precise versus
accurate sizing
Whenevaluatingsizing,considertwometrics:
Precision(reproducibility)Themeasureoftheabilitytogeneratethesamesize
consistentlyforagivenfragmentobtainedunderthesameconditions.
AccuracyThemeasureoftheabilitytogeneratefragmentsizesthatarecloseto
theactualsizeasdeterminedbysequencing.
ThesizeofaDNAfragmentisalteredbythedyewithwhichitislabeled,andeach
ThermoFisherScientificdyehasadifferentsize.Therefore,afragmentwithaknown
sizemaybesizeddifferentlywhenrunusingThermoFisherScientificdyesand
instruments.Althoughthissizemaynotbeaccuratewhencomparedtotheactual
size,itwillbeprecisewhencomparedtootherfragmentsrununderthesame
conditions.
Notethefollowing:
Sizingdifferencesbetweenvarioustypesofpolymeraremoreapparentfor
sequences<50basepairs(bp).
Smallerfragments(<50bp)runonPOP7polymeron3730/3730xlinstruments
mayhaveslightlylowersizingprecision.
Relative sizing
Thesizeofafragmentiscalculatedbasedonthesizestandardwithwhichit
comigrates.DyelabeledDNAfragmentscanyielddifferentsizeswhenrunwitha
differentinstrument,polymer,capillaryarraylength,orsizestandardasshownbelow.
Highprecisionisimportantinrelativesizing.
Sample 1
310 instrument
Sample 1
3130 instrument
Guidelines for
consistent sizing
Usethesamesizingmethodforallinjections.
Toverify,checktheanalysismethodintheGeneMapperManagerorintheSize
MatchEditorwindow.
Usethesamesizestandardforallsamplesinarun.Youmayneedtomodifythe
sizestandarddefinitionofindividualsamples.
Toverify,overlaythesizestandardpeaksfromallinjectionsordisplaythesizing
curveforeachsamplefile.
VerifythatalldefinedsizestandardpeaksarepresentintheSizeMatchEditor.
Variablerunconditionscanoccasionallycausesizestandardpeaksnottobe
detected,forexampleifarunistoofast,tooslow,orifthesignalintensityofsome
ofthepeaksistoolow.
UseanAnalysisRangethatincludesallthescans(ordatapoints)where
sizestandardpeaksoccurintherawdataofeachsample.
Toverify,checktheanalysismethodintheGeneMapperManager.
90
GeneMapper Software features
Autoanalysis and
manual analysis
(GeneMapper
Software only)
TheGeneMapperSoftwareprovidestwoanalysisoptions:
AutoanalysisThesoftwareappliesananalysismethod,sizestandard,and
(optionally)paneltothefragmentanalysisfilesimmediatelyafterDataCollection
Softwarecollectsthedatafromtheinstrument.Theanalysissettingsaresavedin
theGeneMapperSoftwareproject.Youcanreviewtheanalyzeddatausing
GeneMapperSoftware.
ManualAnalysisYouobtainthefragmentanalysisfilesfromthecomputer
connectedtotheinstrument.IftheDataCollectionSoftwareandthe
GeneMapperSoftwareareinstalledonthesamecomputer,youcanimportthe
datafilesintotheGeneMapperSoftware.IftheDataCollectionSoftwareandthe
GeneMapperSoftwareareinstalledondifferentcomputers,moveorcopythe
filestoanothercomputerthathasGeneMapperSoftwareinstalled.Toperform
analysis,youmanuallyapplytheanalysisparameterstothefragmentanalysis
filesintheGeneMapperSoftware.
GeneMapper Software features

Feature
Description
Autoanalysis
Yes, with the corresponding Data Collection Software version
Applications
Amplified fragment length polymorphism (AFLP), loss of heterozygosity (LOH), microsatellite

genotyping, and SNaPshot genotyping.
Analysis of large fragments (~1200 bp), BAC fingerprinting, genetic fingerprinting, multilocus
variant analysis (MLVA), Fragile X assays, biodefense,
T/B-cell clonality assay, bird sex identification, microsatellites, VNTRs, T-RFLP.
Regulatory
compliance
Security and audit features to help users meet 21 CFR Part 11 requirements
Report
Report Manager tools for customized report generation

Customization of the project auto-saving frequency
Analysis
Definition of a linearity range in the analysis methods

Process Quality Values (PQVs) for automated evaluation
Sizing methods
Least Squares, Cubic Spline, Local Southern, and Global Southern
Instrument
software
Supports data generated on 3500 Series, 3730 Series, 3130 Series, and 310 instruments.
Use environment
Multiuser, client-server deployment
GeneMapper Software v4.1 and later includes the ability to record and reapply the Size
Standard Normalization factor calculated in 3500 Series Data Collection Software
Remote auto-analysis and command line operation
Support
Fully supported by Thermo Fisher Scientific
91
Peak Scanner Software Features
Peak Scanner Software Features

Overview
PeakScannerSoftwareisanucleicacidsizingsoftwarethatidentifiespeaksand
fragmentsizesforapplicationspecificcapillaryelectrophoresisassays.Thissoftware
allowsyoutoannotatedatawithfunctionssuchaslabeling,merging,andsplitting
peaks.Thesoftwarestoresalleditingandanalysisdataintheoriginal.fsadatafiles
generatedonThermoFisherScientificgeneticanalysisinstruments.
PeakScannerSoftwareisavailablefreeofchargeonwww.lifetechnologies.com.
Note: ThermoFisherScientificdoesnotsupportPeakScannerSoftware.
Usethissoftwarewithdatageneratedon3730Series,3130 Series,and310instruments.
Itisnotcompatiblewithdatageneratedonthe3500Seriesinstrument,whichperforms
fragmentsizingduringdatacollection.
Features
Importandanalyzefragmentanalysissamplefiles(.fsa)fromallcurrently
supportedThermoFisherScientificgeneticanalyzers
Analyzeddata(sizinginformation)iswrittenbacktothesamplefiles(.fsa)
Abilitytoorganizethesamplefilesinaproject
Simultaneousviewingofrawandanalyzeddata
Largefragmentsizingupto1200bp
Abilitytodefinetheexpectedlinearrangeinlargefragmentsizestandardswhere
nonlinearitymightbeexpected
Expandedfeaturesetforeditingpeaksthatincludeslabeling,merging,and
splittingpeaks
Customizablesizingtable
Abilitytooverlaysizingcurvesonanalyzeddata
Abilitytodisplayandprintplotsinthumbnailview
Lightweightsoftwareapplicationwitheasyinstallation
Abilitytoarchiveprojectswithsamplefilesandassociatedreferencedata
(analysismethods,sizestandardsandsoon)fordatasharingpurposes
92
Workflow
Workflow
Step
Set up a run file
Define analysis
parameters
GeneMapper Software
1. Create a new project.
Peak Scanner Software
2. Click to add samples to the project.
Import Sample files (drag and drop or add

files function.)
In the Samples tab, specify the analysis

parameters for the samples in the project:
Choose appropriate size standard and

analysis method.
1. In the Analysis Method column, select or

create an analysis method depending on
the application used (AFLP,
Microsatellite, SNaPshot)
2. In the Panel column, select None or
Project Specific Panel.
3. In the Size Standard column, select the
appropriate size standard.
Analyze
Analyze the data by clicking the green arrow

to analyze the samples in the project.
Analyze the data by clicking the green arrow

to analyze the samples in the project.
Review the data
1. (Optional) In the Genotypes tab, review the

Process Quality Value (PQV) columns (BD,
BIN, CC, LPH, OBA, OS, PHR, SHP, SP,
SPA, SPU, and XTLK).
1. View sizes in sizing table and label and/or

edit peaks.
2. Review the size quality and sizing data: In

the Samples tab, examine the Size Quality
(SQ) scores and the size standards.
3. Modify sizing or analysis parameters if
necessary.
2. Save project and print or export results if

necessary.
3. Check sizing quality.
4. (Optional) Modify sizing or analysis
parameters if necessary.
4. Display the samples and genotypes plots.

5. (Optional) Save/print/export the results.
ForalistoftheGeneMapperSoftwaredocumentsavailable,see,Documentation
andSupportonpage 199.
93
GeneMapper Software peak detection settings

Peak Amplitude
Thresholds
Onlypeakswithheightsthatexceedthepeakamplitudethresholdvaluesforadye
coloraredetected.
Smoothing
Smoothingoptimizespeaksizeandcanreducethenumberoffalsepeaksdetected:
None(default)appliesnosmoothing.Noneisusefulifthedatadisplaysharp,
narrowpeaksofinterest.
Lightprovidesthebestresultsfortypicaldata.Lightsmoothingslightlyreduces
peakheightintheelectropherogram.Itdoesnotaffecttabulardata.
Heavyisusefulfordatafromslowerrunsthatdisplaybroadpeaksortoavoidthe
detectionofsharpedges.Thisselectionmayreducepeaksizeoreliminatenarrow
peaksintheelectropherogram.Itdoesnotaffecttabulardata.
Baseline Window
TheBaselineWindowadjuststhebaselinesofalldetecteddyecolorstothesamelevel
foranimprovedcomparisonofrelativesignalintensityandhelpstoeliminatenoise
fromthebaseline.
IftheBaselineWindowvalueistoolow,thebaselineapproachesthepeaksand
thedatadisplayshorterpeaks.
IftheBaselineWindowvalueistoohigh,thebaselineistoolowandthedata
displayelevatedandpossiblynotbaselineresolvedpeaks.
Min. Peak Half

Width
TheMin.PeakHalfWidthsettingspecifiesthesmallestfullwidthathalfmaximumfor
peakdetection.
Usealowvalueifthedatadisplaynarrowpeaks.
Ifthevalueishigh,noisespikesareignored.
Polynomial Degree
and Peak Window
Size parameters
UsethePolynomialDegreeandthePeakWindowSizesettingstoadjustthesensitivity
ofthepeakdetection.Youcanadjusttheseparameterstodetectasinglebasepair
differencewhileminimizingthedetectionofshouldereffectsornoise.
Sensitivityincreaseswithlargerpolynomialdegreevaluesandsmallerwindowsize
values.Conversely,sensitivitydecreaseswithsmallerpolynomialdegreevaluesand
largerwindowsizevalues.
Thepeakdetectorcalculatesthefirstderivativeofapolynomialcurvefittedtothedata
withinawindowthatiscenteredoneachdatapointintheanalysisrange.
Usingcurveswithlargerpolynomialdegreevaluesallowsthecurvetomoreclosely
approximatethesignaland,therefore,thepeakdetectorcapturesmorepeakstructure
intheelectropherogram.
Thepeakwindowsizesetsthewidth(indatapoints)ofthewindowtowhichthe
polynomialcurveisfittedtodata.Higherpeakwindowsizevaluessmoothoutthe
polynomialcurve,whichlimitsthestructurebeingdetected.Smallerwindowsize
valuesallowacurvetobetterfittheunderlyingdata.
94
Function
Effects of varying
the Polynomial
Degree
Polynomial Degree Value
Window Size Value
Increase sensitivity
Higher
Lower
Decrease sensitivity
Lower
Higher
Thefigurebelowshowspeaksdetectedwithawindowsizeof15datapointsanda
polynomialcurveofdegree2(green),3(red),and4(black).Thediamondsrepresenta
detectedpeakusingtherespectivepolynomialcurves.
Notethatthesmallertrailingpeakisnotdetectedusingadegreeof2(green).Asthe
peakdetectionwindowisappliedtoeachdatapointacrossthedisplayedregion,a
polynomialcurveofdegree2couldnotbefittedtotheunderlyingdatatodetectits
structure.
Polynomial curve of degree 4

(black)
(red)
(green)
95
Effects of
Increasing the
Window Size Value
Inthefigurebelow,bothpolynomialcurveshaveadegreeof3andthewindowsize
valuewasincreasedfrom15(red)to31(black)datapoints.
Asthecubicpolynomialisstretchedtofitthedatainthelargerwindowsize,the
polynomialcurvebecomessmoother.Notethatthestructureofthesmallertrailing
peakisnolongerdetectedasadistinctpeakfromtheadjacentlargerpeaktotheright.
Window size value of 31 (black)

Window size value of 15 (red)
96
GeneMapper Software peak start and end settings
GeneMapper Software peak start and end settings

TheSlopeThresholdforPeakStartandSlopeThresholdforPeakEndparameters
adjustthestartandendpointsofapeak.
Thevaluesassignedtotheseparameterscanbeusedtobetterpositionthestartand
endpointsofanasymmetricalpeak,orapoorlyresolvedshoulderingpeaktomore
accuratelyreflectthepeakpositionandarea.
Ingeneral,fromlefttoright,theslopeofapeakincreasesfromthebaselineuptothe
apex.Fromtheapexdowntothebaseline,theslopedecreasesnegativelyuntilit
returnstozeroatthebaseline(seethefigurebelow).
Apex
Baseline
Increasingly
positive slope
(+)
0
Increasingly
negative slope
()
0
Notethefollowing:
Fortypicalorsymmetricalpeaks,useavalueofzero.
Forasymmetricalpeaks,selectvaluesotherthanzerotobetterreflectthe
beginningandendpoints.
Avalueofzerodoesnotaffectthesizingaccuracyorprecisionofanasymmetrical
peak.
Note: Thesizeofadetectedpeakisthecalculatedapexbetweenthestartandend
pointsofapeak.Peaksizedoesnotchangebasedonstartandendsettings.
To move the
Then
Start point of a peak closer

to its apex
Change the Slope Threshold for Peak Start

value from zero to a positive number.
End point of a peak closer to

its apex
Change the Slope Threshold for Peak End

value to a more negative number.
Example
97
How the GeneMapper Software performs sizing

TheGeneMapperSoftwareandthePeakScannerSoftwareusesettingsinthe
analysismethodtosizesamples.
Thissectionprovidesabriefdescriptionofthesizingprocess.Formoreinformation,
seetheGeneMapperSoftwareReferenceandTroubleshootingGuide(Pub.no.4403673).
Size-standard
definitions
Duringsizing,thesoftwarecomparesthesizeofobservedfragmentsforthesize
standardinthesampletotheexpectedfragmentsizeslistedinthesizestandard
definitionusedforanalysis.
TheGeneMapperSoftwareincludesseveralsizestandarddefinitions.Youcanalso
createyourownsizestandarddefinitionfilesordownloadupdatedsizestandard
definitionsfromourwebsite.
Datafrom3500Seriesinstrumentscanbeanalyzedwithasizestandarddefinitionthat
specifiesnormalizationifthedatawascollectedwithanormalizationstandard.
Step 1: Size
matching
Sizematchingusesratiomatching,basedonrelativeheightanddistanceof
neighboringpeaks.Itthenderivesqualityvaluesstatisticallybyexaminingthe
similaritybetweenthetheoretical(fromthesizestandarddefinition)andactual
(observed)fragmentpatterns(seethefigureonthenextpage).
Tocompletethisstepsuccessfully,theanalysissoftwaremustmatchatleastthree
peaks.
Thesoftwareignoresanomalouspeaksthatdonotmatchtheexpectedpatterns.The
softwareconstructsabestfitcurveusingthedatapointsofeachsizestandard
fragmentdetected.Acomparisonbetweenthesizescalculatedfromthebestfitcurve
andthematchedpeaksfromthesizestandarddefinitionusingthearrayofnumbersis
performed.Sizematching(andsubsequentsizing)failsifsignificantdifferencesin
peakpatternsarefound,ifnomatchcanbemadebasedontheexpectedpatterns,orif
allpeaksarenotfound.
Becausethesoftwareusesratiomatching(looksfortheexpectednumberofallelesand
expectedpeakpatternsinsteadofspecificdatapoints),itisnotnecessarytodefinenew
sizestandarddefinitionstoaccommodatemigrationshifts.
98
Determines the expected peak spacing and height ratios

Uses the list of sizes from the Size Standard definition.
Note: The values used are for example only and do not reflect typical size standard values.
Base
pair
50
100
x
200
400
2x
4x
Evaluates peaks in the size standard data

Ignores peaks that do not meet expected pattern (dotted peak).
Base
pair
50
100
x
Data
100
point
200
2x
200
400
Base
pair
50
less than
4x
100
x
400
800
Data
100
point
200
2x
200
400
4x
400
800
Plots the sizing curve

Uses peaks that meet expected pattern
500
Base pair
400
300
200
100
0
0
100 200 300 400 500 600 700 800 900
Data point
99
GeneMapper Software sizing methods
Step 2: Sizing curve

and sizing
Factors that affect
sizing
Togeneratethesizecallingcurve,thesoftwareplotstheactualdatapointsofthesize
standardagainsttheexpectedsizeofeachsizestandardpeak.Thesizecallingmethod
determineshowthesizecallingcurveisgeneratedandusedtosizeeachsample.
Thesizingmethod,sizestandarddefinition,orsizestandardusedtogeneratethe
sizingcurve
Welltoreadortimetoreaddifferences
Electrophoresisconditions,suchasruntemperature,voltage,orthedenaturing
abilityoftheseparationmatrix
Polymertype(POP4,POP6,POP7)andconcentration
Capillarylength(22cm,36cm,or50cm)
Instrumentmodelduetodifferencesininstrumentconfiguration

Thesizingmethodsavailableinclassicandadvancedmodesintheanalysismethodof
theGeneMapperSoftwareare:
LeastSquares
CubicSplineInterpolation
LocalSouthern
GlobalSouthern
Globalmethods,whichgeneratethebestfitcurvefromallmatchedfragmentsinthe
sizestandard,arelessaffectedthanlocalmethodsbyanomaliesintheruntimesof
singlesizestandardfragments.Doesnotnormalizecapillarytocapillary.
Localmethods,whichgeneratethebestfitcurvefromnearbysizestandarddata
points,arelessaffectedbychangesintheelectrophoresisconditionsorintheanalysis
range.(Achangeintheanalysisrangechangesthesubsetofsizestandardfragments
thatisavailableforgeneratingthesizingcurve.)Normalizescapillarytocapillary.
Least Squares
method
100
Advantages
BothLeastSquaresmethods(2ndOrderand3rdOrder)useregressionanalysisto
buildabestfitsizingcurve.Thiscurvecompensatesforanyfragmentsthatmayrun
anomalously.Asaresult,thismethodnormallyresultsintheleastamountofdeviation
forallthefragments,includingthesizestandardsandthesamples.Dependingon
whetheryouchoosethe2ndor3rdOrderLeastSquaresMethodintheAnalysis
Parametersdialogbox,theresultingsizecurveiseitheraquadraticoracubicfunction.
Thesoftwareusestheknownstandardfragmentsandtheassociateddatapointsto
produceasizingcurvebasedonMultipleLinearRegression.
Inthefollowingfigures,youcanseethatinnearlyallinstancesthemobilityofan
individualDNAfragmentiscoincidentwiththebestcurvefitoftheentiredataset.
Stateddifferently,themobilityofmostDNAfragmentsisstrictlylengthdependent.
Thismethodautomaticallycompensatesforfragmentsthatrunanomalously.
GeneMapperSoftwarecalculatesabestfitleastsquarescurveforallsamples,
regardlessofthesizingmethodyouchoose.ThecurveisblackintheStandardSizing
Curvewindow.
Note: AllofthegraphsinthissectionweregeneratedusingGeneScanSoftware
v3.7.1.TheseresultsaresimilartoresultsobtainedwhenyouuseGeneMapper
Softwarev3.5andhigher.
2nd-Order Least Squares sizing curve
Cubic Spline
Interpolation
method
3rd-Order Least Squares sizing curve
TheCubicSplinemethodforcesthesizingcurvethroughalltheknownpointsofthe
selectedsizestandard.Althoughthisenforcementproducesexactresultsforthevalues
ofthestandardsthemselves,itdoesnotcompensateforstandardfragmentsthatmay
runanomalously.
Cubic Spline interpolation sizing curve
101
Possible local sizing inaccuracy

MobilityofanyDNAfragmentcanbeaffectedbyitssequence,andbysecondaryand
tertiarystructureformation.Ifanyinternalsizestandardfragmenthasanomalous
mobility,theCubicSplinemethodmayexhibitlocalsizinginaccuracy.Forexample:
Assumethatastandardfragmentiscloseinmolecularlengthtoanunknownsample
fragment.Assumefurtherthatthestandardfragmentrunsanomalously.TheCubic
Splinemethodassignstheofficialvaluetothisstandardfragment,eventhoughitmay
beslightlyincorrect.Thesizeoftheunknownfragmentisthenlikelytobecalculated
incorrectlyaswell.
Note: Thismethoddoesnotdeterminetheamountofsizingaccuracyerror.
Local Southern
method
TheLocalSouthernmethoddeterminesthesizesoffragmentsbyusingthereciprocal
relationshipbetweenfragmentlengthandmobility,asdescribedbyE.M.Southern
(1979).
IMPORTANT! FortheLocalSouthernMethodtowork,youmusthaveatleasttwo
sizestandardfragmentssmallerthanyoursmallestunknownfragmentandtwo
sizestandardfragmentslargerthanyourlargestunknownfragment.Ifyoudonot,a
secondorderleastsquarescurveextrapolationwillbeusedtoderivethesizecurve,
insteadofthemethodspecifiedintheanalysismethod.
Local Southern sizing curve
Local Southern Method equation

L=[c/(mm0)]+L0
Theequationattemptstodescribethereciprocalrelationshipbetweenthemobility,m,
andthelength,L0,ofthestandardfragments.
How the Local Southern method works

Thismethod,whichissimilartotheCubicSplinemethod,usesthefourfragments
closestinsizetotheunknownfragmenttodetermineabestfitlinevalue.Onlythe
regionofthesizestandardnearthefragmentofunknownlengthisanalyzed.
Note: Sizeestimatesmaybeinaccurateifanyofthesizestandardfragmentsrun
anomalously.
102
ThisishowtheLocalSouthernmethodworks:
1. Thefittingconstantsofthecurvearecalculatedforeachgroupofthree
neighboringpointsonthestandard.Aseparatecurveiscreatedforeachsetof
threepoints.
2. Acurveisthencreatedbyusingthreestandardpoints(twopointsbelowandone
pointabovethefragment)andafragmentsizeisdetermined.
3. Anothercurveiscreatedbylookingatanadditionalsetofthreepoints(onepoint
belowandtwopointsabovethefragment)andanothervalueisassigned.
4. Thetwosizevaluesareaveragedtodeterminetheunknownfragmentlength.
Global Southern
method
ThismethodissimilartotheLeastSquaresmethodinthatitcompensatesforstandard
fragmentsthatmayrunanomalously.Themethodcreatesabestfitlinethroughallthe
availablepoints,andthenusesvaluesfoundonthatlinetocalculatethefragment
values.
Global Southern sizing curve
Global Southern method equations

Equation
Description
Attempts to describe the reciprocal
relationship between the mobility, m, and the
length, L0, of the standard fragments.
The fitting constants L0, m0, and c are
calculated by a least-squares fit to minimize
the left side quantity.
How the Global Southern method works

Allpointsinthestandardareweightedequallyandthecurveisnotconstrainedto
passthroughanyspecificpoint.Thesoftwarecananalyzealargerangeoffragment
sizeswiththismethod.
Forbestresults,useasizestandardthathasatleasttwopeakssmallerthanthe
smallestfragmentofinterestandatleasttwopeakslargerthanthelargestfragmentof
interest.
103
Evaluating data quality

Note: Fordetailedinformationonqualityvaluedetermination,seetheGeneMapper
SoftwareReferenceandTroubleshootingGuidev4.1(Pub.no.4403673).
Examining PQVs
TheGeneMapperSoftwaredisplaysProcessQualityValues(PQVs)intheSamplesor
GenotypestaboftheProjectwindow.
Symbol
Default
Range
Definition
Pass: The sample or genotype passed the PQV test.
0.75 to 1.0
Check: A possible problem exists for the sample or

genotype.
0.25 to 0.75
Low Quality/Fail: There is a strong possibility that a

problem exists for the sample or genotype.
0.0 to 0.25
ReviewtheSQ(SizingQuality)andGQ(GenotypeQuality)resultsforeachsample.
ManyofthePQVscanaffecttheGQresult.
Note: IftheSQPQVis
.
,thesampleisnotsizedorgenotyped,andtheGQPQVis
Werecommendexaminingallsamplesthatproduce
SQflags.
(Check)or
(LowQuality)
ForinformationonconfiguringandinterpretingPQVs,refertotheGeneMapper
SoftwareReferenceandTroubleshootingGuidev4.1(Pub.no.4403673).
ForinformationontroubleshootingSQ
page153.
Criteria for a good

electropherogram
results,seeCheckingdataqualityon
Figure20illustratesanelectropherogramthatmeetsthefollowingcriteria:
Peakheightsare50RFU(peaks<50RFUareconsideredtobenoise).
Idealpeakheightsare:
3500Seriesinstruments:175RFU
3730Series,3130Series,and310instruments:150RFU
Peaksaresharpwithnoshouldersorsplits.
Thepeakscorrespondingtodifferentcolordyesmaynotbeofequalintensity,but
thedataforthelessintensecolorsshouldbeclearlyresolvableathigher
magnification.
Allexpectedpeaksaredetected.
Peaksaresizedproperly(seeSizestandardsonpage42).
Ifyouaregenotyping,samplesareaccuratelygenotyped.
Resultsarereproducible.
104
Figure20 Example of a good electropherogram
Ifelectropherogramsdonotmeetthecriteriaabove,seeChapter11,Troubleshooting
onpage 151.
Examining peak
definitions
ToexaminehowGeneMapperSoftwarehasdefinedapeak,select
ViewShowPeakPositions.Thepeakpositions,includingthebeginning,apex,and
endofeachpeak,aretickmarkedintheelectropherogram.
Comparing data
UsethesameGeneScansizestandardlabeledwiththesamedyeforallsamples
inasinglestudy.
Comparepeakareas,heights,andsizesinnucleotidebasesonlyiffragmentsare
labeledwiththesamedye.Formoreinformation,seePreciseversusaccurate
sizingonpage90andRelativesizingonpage90.
Compareonlydatathatiscollectedunderthesameconditions(capillaryarray
length,polymertypeandelectrophoreticrunconditions)forthesamestudy
becausetheseconditionsaffecttherelativesizeofthefragment.
105
106
Microsatellite Analysis
Overviewofmicrosatelliteanalysis ..................................... 107
Applications ........................................................ 110
Instrumentandconsumablerecommendations .......................... 111
Experimentandprimerdesignrecommendations........................ 111
Workflow .......................................................... 112
Dataanalysis ........................................................ 112
Commonproblemswithmicrosatelliteanalysis.......................... 113
Identifyingstutterproductsinmicrosatelliteanalysis ..................... 113
Formoreinformation................................................. 118
Overview of microsatellite analysis

Microsatellitemarkers,alsocalledshorttandemrepeat(STR)markers,are
polymorphicDNAlocithatcontainarepeatednucleotidesequence.Eachrepeatunit
canbe2to7nucleotidesinlength,andallelesdifferbythenumberofrepeats.The
numberofnucleotidesperrepeatunitisthesameforamajorityofrepeatswithina
microsatellitelocus.
Microsatellitemarkersarealsoknownas:
Shorttandemrepeats(STRs)
Simplesequencerepeats(SSRs)
Variablenumbertandemrepeats(VNTRs)
107
Chapter6 Microsatellite Analysis

Principle of the
analysis
Microsatelliteanalysisistheseparationoffluorescentlylabeledfragmentsusing
forwardandreverseprimersanddeterminationoftherelativesizeofthefragments.
APCRprimerpairconsistsoftwooligonucleotides(forwardandreverseprimers),
typically15to30nucleotideslong.Eachprimerhybridizestoitsrespective
complementarystrandoftheDNAtemplatesuchthattheprimerpairflanksthe
regionofinterest.Basedontheapplication,oneorbothoftheprimersmaybelabeled
withafluorescentdye.
Thenumberofrepeatunitsatamicrosatellitelocusmaydiffer,soallelesofmany
differentlengthsarepossibleateachlocus.Themicrosatellitemarkerinthefigure
belowcontainsadinucleotiderepeat.WhenPCRisperformedusingprimersthatflank
theregionofinterest,PCRfragmentsofdifferentsizesaregeneratedbasedonthe
lengthofthedinucleotiderepeat.
Figure21 Different repeats lead to PCR fragments of different length (arrows indicate forward
and reverse primers)
Advantages of
using
microsatellite
markers (loci) in
genetic studies
Severalfeaturesofmicrosatellitesandtheircorrespondingsetofallelesmakethem
idealforuseingeneticstudies:
Theyarepresentinlargenumbers.
Theyarerelativelyevenlyspacedthroughoutthegenomeandoftenphysically
situatednearorwithingenes.
Theyshowavarying,butrelativelyhighmutationraterelativeto
nonmicrosatelliteloci:
MitochondrialDNAevolves5to10timesfasterthansinglecopynuclear
DNA
Microsatellitesevolve100to1000timesfasterthansinglecopynuclearDNA
Themutationrateofmicrosatellitelociis102to106eventsperlocusper
generation(Wan,etal.,2004).Therateisbelievedtobedifferentdependingonthe
numberofnucleotidesintherepeatedunit(EckertandHile,2009).
108
TheirallelesareinheritedinaMendelianmannerandarestableovermultiple
generations.
Theirallelescanbeuniquetospecificpopulations.
Detaileddataonallelicvariation,numberofrepeats,andallelicfrequenciesare
widelyavailableforalargenumberofmicrosatellitemarkers.
Thesmallsizeofmicrosatellitelociimprovesthechanceofobtainingaresult,
particularlyforsamplescontainingverylowamountsofDNAand/ordegraded
DNA.
Thesmallsizerangeofmicrosatellitelocimakesthemidealcandidatesfor
coamplificationwhilekeepingallamplifiedallelessmallerthan350basepairs.
ManymicrosatellitelocicanthereforebetypedfromasinglePCR.
Microsatellitealleleshavediscretesizes,allowingforsimplifiedinterpretationof
results.
PCRbasedtestsarerapid,givingresultsin24hoursorless.
PCRbasedtestsareeasytostandardizeandautomate,ensuringreproducible
results.
Microsatellite
motifs and
distribution
STRstypicallycontain2to7nucleotiderepeats.
VNTRscontain10to100nucleotiderepeatingmotifs.
Figure22 STRs compared to VNTRs
Often,thelengthoftherepeatingunitcorrelateswithitsfrequencywithinagenome.
Forexample,inthehumangenome,mononucleotiderepeatsarethemostcommon
formofmicrosatellitesfound,andpentanucleotideandhexanucleotiderepeatsarethe
leastcommon(Ellegren,2004).
However,thefrequencyofarepeatingunitcanvaryacrossaparticularchromosome
asshowninthefollowingfigure.
109

Applications
Figure23 SSR density in exonic, intronic and intergenic regions on individual human chromosomes: (a) monomers (b)
dimers; (c) trimers; (d) tetramers; (e) pentamers; (f) hexamers. Blue bars, exons; red bars, introns; yellow bars, intergenic
regions (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC151303/figure/F2/ Copyright 2003, Subramanian et al.; licensee
BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.)
Applications
Thelargeselectionofhighlyinformativemarkershasmademicrosatelliteanalysisa
widelyacceptedtoolforthefollowingtypesofstudies:
Linkagemappingstudies
Associationstudies
Populationstudies
Parentageanalysis
Breeding
Custommicrosatelliteassaysareoftenusedfor:
Cancerprogressionanalysis
Phylogeneticstudies
Genomescansforanorganismwherecommercialmarkerpanelsarenotavailable
Populationgeneticsstudies
Paternitytesting
Parentageanalysisforselectivebreeding
110

Instrument and consumable recommendations
Instrument and consumable recommendations

ThisisaThermoFisherScientificsupportedprotocol.
Thermalcycler:2720,Veriti,orGeneAmp9700
Geneticanalyzer:3500 Series,3130 Series,or310instrument
Polymerandcapillaryarray:seeRunmodulesonpage69forthepolymerand
capillaryarraylengthcombinationssupportedoneachinstrument
600LIZSizeStandard
DS33G5dyeset
AmpliTaqandAmpliTaqGoldDNAPolymerasesaretypicallyusedfor
microsatelliteanalysis.LikeotherDNApolymerases,thesepolymerasesmay
catalyzetheadditionofasinglenucleotide(usuallyA,adenosine)tothe3endsof
thetwostrandsofadoublestrandedDNAfragment.Formoreinformation,see
Additionof3AnucleotidebyTaqpolymeraseonpage33andWitmeretal.,
2003.
IMPORTANT! Throughoutasetofexperiments,usethesameequipment,run
parameters,polymers,dyes,andsoon.Consistentconditionsarerequiredtoavoid
mobilityshiftsthatinterferewithaccurateinterpretationofdata.
Experiment and primer design recommendations

Identifythemarkersforyourstudybyexaminingtheexistingscientificliterature
foraspecificmarkerorcrossspeciesmarker,orbyfollowingamicrosatellite
developmentprotocol(FleischerandLowe,1995;Kandpalet.al.,1994).
Thediscoveryandrandomnamingofnewmicrosatellitemarkersacrossdifferent
organismsatmultipleinstitutionshasledtoinconsistentnomenclaturesfor
microsatellites.Formoreinformationonnomenclaturestandardsforspecific
genomes,gotothenomenclaturewebsiteoftheinstitution,afewofwhichare
listedbelow.
Human:https://iris.ucl.ac.uk
Rat/mouse:http://rgd.mcw.edu/
Fly:http://flybase.org/
Designprimerssotherangeofampliconlengthsacrossmarkersinthestudy
spansiswithinthesizestandardfragmentrange,withtwosizestandardpeaks
precedingthesmallestfragmentofinterestandtwosizestandardpeaks
followingthelargestfragmentofinterest.
Use5endlabeledprimers.Thesuccessofmicrosatelliteanalysisdependsupon
theabilitytodetectsmallmobilitydifferences.Thereproduciblesizingandsharp
peaksobtainedwhenusingthe5endlabelingmethodarecrucialtothesuccess
ofthisapplication.
Ifyouplantomultiplex,designprimerswithsimilarannealing
temperatures ~60C.
Usereverseprimertailingononeprimerineachsetofprimerstohelp
differentiatebetweenpeaksmadebytheforwardandreverseDNAstrandsand
topromoteAaddition.SeeTailonpage 34formoreinformation.
111

Workflow
BasedonsampleDNAconcentration,robustnessofthePCR,and/orpeakheights
observedincapillaryelectrophoresis,determinewhetheryouneedtodilutethe
PCRproducts.Dilutionscanrangefromundilutedto1:20inwater.Youcanpool
thedilutedPCRproductsifdesired.
DyelabeledPCRproductsmustbemixedindifferentratiosbecauseeachdyehas
aslightlydifferentfluorescencesignalstrength(seeEmissionandabsorption
(excitation)wavelengthsandrelativeintensitiesonpage38).
Toavoidinaccuraciesassociatedwithpipettingsmallvolumes,prepareamaster
mixofreagents.Preparesufficientmastermixforatleastoneextrareaction
volume.
Storethemastermixinthedarkat2to6Cforupto1month,orat15to25C
forlonger.
Atypicalreactionmayinclude:1LofeachPCRproductand0.5Lofthe
GeneScansizestandardin8.5LofHiDiformamide(fordenaturing
applications)ordistilled,deionizedwater(fornondenaturingapplications).
Mastermixreagentsareoptimizedforcapillaryelectrophoresis,anddiffer
dependingonthecapillaryelectrophoresisinstrumentyouuse.
Workflow
1. Selectprimersandsizestandards:
a. Designandorderprimersforamicrosatelliteapplication.
b. OptimizeamplificationconditionswithmicrosatellitemarkersontestDNA.
c. Orderdyelabeledprimers.
2. PCR
3. Capillaryelectrophoresis
4. Dataanalysis
Data analysis
TheGeneMapperSoftwareincludesaMicrosatelliteDefaultanalysismethodthat
youcanuseasastartingpointforanalysis.
Figure24onpage113showsatypicalmicrosatelliteelectropherogramfromthe
GeneMapperSoftware.
Thenumberofrepeatsforagivenlocusmayvary,resultinginallelesofdiffering
lengths.ThefollowingfigureshowstwodifferentFAMdyelabeledhuman
dinucleotideloci(fromtheGeneMapperSoftwaretutorialdataset)fromtwo
individuals.ThetoppanelillustratesaDNAsamplethatishomozygousatbothloci(a
singlemajorpeakisobservedateachlocus),thebottompanelshowsaDNAsample
thatisheterozygousatthesameloci(twomajorpeaksareobservedateachlocus).
112

Common problems with microsatellite analysis
Figure24 Example of microsatellite analysis of two samples by capillary electrophoresis.

Samples have different genotypes as shown by the different peaks for the same marker.
Common problems with microsatellite analysis

Themostcommonlyencounteredproblemsduringmicrosatelliteanalysisare:
Poorornonspecificamplification.SeeOptimizingPCRonpage55andPCR
troubleshootingonpage176.
Incomplete3 Anucleotideaddition.SeeIncomplete3Anucleotideaddition
onpage33.
Stutter.Seethenextsection.
SeeChapter11,Troubleshootingonpage151formoreinformationonstutterpeaks
andplusAproducts.
Identifying stutter products in microsatellite analysis

Overview
DuringthePCRamplificationofdi,tri,andtetranucleotidemicrosatelliteloci,minor
productsthatare1to4repeatunitsshorterthanthemainalleleareproduced.The
minorproductpeaksarereferredtoasstutterpeaks.Stutterpeaksmaybecausedby
polymeraseslippageduringelongation(HaugeandLitt,2003;MurrayandLai,2003).
113

Stutterpeaksappearasmultiplelowerpeaksthatprecedethetrueallelepeak.These
stutterpeaksdifferinsizefromthetrueallelepeakbymultiplesofthelengthofthe
repeatunit.Thenumberofpeaksandtheirintensitiesareproportionaltothelengthof
therepeatandthenumberofrepeatsinthePCRproduct(Shindeet.al.1993).Shorter
repeatunits(diortri,forex.)generatemorestutter,anddinucleotiderepeatstendto
generatemorestutterpeaksthantrinucleotiderepeats.
Stutterpeakscanalsobecausedbyoffscaledata.Formoreinformation,see
Evaluatingdatawithstutteronpage117.
GeneMapperSoftwareisoptimizedtofilteroutstutterpeaks.
Estimating the
amount of stutter
Youcanestimatethepercentstutterbycalculatingtheratioofthecombinedheightsof
thestutterpeakswiththeheightofthetrueallelepeak.Notethefollowing:
Thelongertherepeatunit,thelessstutterproductproduced.Formicrosatellite
lociwiththesamenumberofrepeatunits,thepercentstutterisgreaterfor
dinucleotidemicrosatellitelocithanitisfortrinucleotidemicrosatelliteloci,and
soon(Walshetal.,1996).
Thefigurebelowillustratesthegreaterstutterindinucleotide(left)ascompared
totetranucleotide(right)repeatloci.Eachlocusishomozygous,withthelargest
peakineachfigurerepresentingthetrueallele.
Thepercentstutterincreaseswithincreasingallelelength(thatis,withincreasing
numberofrepeatunits).However,ifsomeoftherepeatsarepartialrepeats,you
maynotseetheproportionateincreaseinpercentstutter.
114

Figure25 Stutter percentages for the FGA and TH01 loci. (Black data points indicate loci labeled
with NED dye.)
Dinucleotide
repeats
Successfulamplificationofdinucleotiderepeatmarkersyieldsallelepeaksand
associatedstutterpeakswithinamaximumrangeofeightbasepairsfromtheallele
peak.Inaddition,thenumberofallelepeaksdependsonwhethertheindividualtested
isaheterozygoteorhomozygote.
Dinucleotide repeats in a homozygous individual

TheGeneMapperSoftwareelectropherogramofadinucleotiderepeatmarkerfroma
homozygousindividual(190bp,190bp)isshowninthefollowingfigure.
Figure26 Stutter peaks in a dinucleotide repeat electropherogram (homozygote)
115

Thepeaksat188bp,186bp,and184bpshowthetypical2bpstutterpatternseenwith
dinucleotiderepeats.Theyrepresentthe2bp,4bp,and6bpstutterpeaksfromthe
true190bptrueallelepeak.
Dinucleotide repeats in a heterozygous individual (8 bp)

TheGeneMapperSoftwareelectropherogramofadinucleotiderepeatmarkerfroma
heterozygousindividual(139bp,147bp)isshowninthefollowingfigure.Allelesizes
differby8bp.
The2bpstutterpeaktotheleftofeachallelepeakisalwaysoflowerintensitythanthe
allelepeakitself.Thelarger147bpallelepeakisoflowerintensitythanthesmaller
139bpallele.Inheterozygotes,thehighermolecularweightallele(thatis,theallele
peakfurthertotherightinelectropherograms)oftenproducesafluorescencesignalof
lowerintensitythanthelowermolecularweightallele,suggestingalessefficient
amplificationofthelargerfragment.Thisphenomenoncouldalsobecausedby
preferentialinjectionofthesmallerfragments.
Figure27 Stutter peaks in a dinucleotide repeat electropherogram (heterozygote 8 bp)

TheGeneMapperSoftwareelectropherogramfromadinucleotiderepeatmarkerofa
differby4bp.
Figure28 Stutter peaks in a dinucleotide repeat electropherogram (heterozygote 4 bp)
116
Whenthedifferencebetweentheallelesizesis4bp,ashiftoccursintheheightratio
betweenthetwoallelepeaks(comparethetwoprecedingfigures).Thefluorescence
signalfromthe4bpstutterofthe189bpalleleisaddedtothesignalfromthe185bp
allele.

TheGeneMapperSoftwareelectropherogramfromadinucleotiderepeatmarkerofa
differby2bp.
Figure29 Stutter peaks in a dinucleotide repeat electropherogram (heterozygote 2bp)
Stutter from
218 bp peak is
added to the
216 bp peak
Stutter band
Whenthedifferencebetweentheallelesizesis2bp,thefluorescencesignalfromthe
2bpstutterofthelargerbasepairalleledoesnotappearasaseparatestutterpeak.It
isaddedtothesignalofthesmallerbasepairallele.
Evaluating data
with stutter
Themultipeakpatternseenwithstutterpeakscancomplicateanalysis,particularlyfor
sampleswithtwoormoreallelesthatarecloseinsize.Forexample,smallpeaksina
positionthatisonerepeatunitsmallerthanthetrueallelecanbeinterpretedeitherasa
stutterpeakorasanalleleinaminorcomponentofamixedsample.Thepossible
presenceofstutterpeaksmakesprecisequantitationespeciallyimportant,toallowthe
GeneMapperSoftwarefilteringalgorithmtointerpretthepeakpatternaccurately.
Thepercentstutterforagivenalleleisreproducibleanddoesnotdependonthe
quantityofinputDNAorthenumberoflociamplifiedduringmultiplexPCR.The
relativereproducibilityofpercentstutterisimportantforafewreasons:
Inmanycases,youcanadjustthePeakAmplitudeThresholdintheanalysis
methodoftheGeneMapperSoftwaretofilteroutstutterpeaksanddetectonly
trueallelepeaks.Foremoreinformation,refertotheGeneMapperSoftware
GettingStartedGuide:MicrosatelliteAnalysis(Pub.no.4403672).
Amplificationswithanabnormallyhighpercentstuttercanindicatemixed
samplesorsomeotherproblemwithPCRamplificationorelectrophoresis.
117

For more information
Is stutter a real
problem?
Stutter,onceunderstood,doesnotposearealproblemformicrosatelliteanalysisand
canaidinallelecallingby:
DistinguishingtrueallelepeaksfromnonspecificPCRproducts.Nonspecific
PCRproductsarenotassociatedwithstutterpeaks.
Identifyingallelesthatfallfaroutsidethereportedallelerange.Thepercent
stutterisoftenspecifictoaparticularlocus.Youcansometimesidentifyalleles
thatfallfaroutsidethepreviouslyreportedrangeonthebasisofpercentstutter.

SeeMicrosatelliteapplicationsonpage200.
118
Single Nucleotide Polymorphism

(SNP) Genotyping
OverviewofSNPgenotyping .......................................... 119
SNaPshotMultiplexSystem.......................................... 120
Overview of SNP genotyping

Overview
ASingleNucleotidePolymorphism(SNP)markerconsistsofasinglebasepairthat
variesintheknownDNAsequence,therebycreatinguptofourallelesorvariationsof
themarker.
...TCGTTGTAGCGCTTAGA...
...AGCAACATCGCTAATCT...
...TCGTTGTAACGCTTAGA...
...AGCAACATTGCTAATCT...
The G-C and A-T

base pairs are two
possible alleles of
this SNP site
SNPmarkersoccurinthehumangenomeatafrequencyofabout1inevery1000bp,
withatotalnumberofover10millionSNPmarkersdistributedevenlyoverthe
3 billionbpsofthehumangenome.Theyhavebeenshowntoberesponsiblefor
differencesingenetictraits,susceptibilitytodisease,andresponsetodrugtherapies.
SNPmarkersareexcellentgeneticmarkerstoconstructhighresolutiongeneticmaps.
SNPmarkerscanbegenotypedbyavarietyofmethods.ThermoFisherScientific
productssupportthefollowingmethods:
Singlebaseextension
ShiftedTerminationAssay(STA)primerextension
Applications (SNP)
SomeapplicationsofSNPgenotypinginclude:
Studyofmutationsimplicatedinvariouscancers
Geneticdiseaseresearch
MitochondrialDNAinvestigations
Scrapiesusceptibilityinsheep
Lossofheterozygosity
Assessperformanceinfoodanimalproduction,
DifferentiatedrugandnondrugformsofCannabis
119
Chapter7 Single Nucleotide Polymorphism (SNP) Genotyping

SNaPshot Multiplex System

Components
TheSNaPshotMultiplexSysteminvestigatesuptotenSNPmarkerssimultaneously
byusingPCRamplification,thendideoxysinglebaseextensionofanunlabeled
primer,andthencapillaryelectrophoresis.Afterelectrophoresisandfluorescence
detection,theallelesofasinglemarkerappearasdifferentcoloredpeaksatroughly
thesamesizeintheelectropherogramplot.Thesizeofthedifferentallelepeakswill
varyslightlyduetodifferencesinmolecularweightofthedyes.
Figure30 Overview of the SNaPshot kit assay
Componentsofthesystemare:
SNaPshotMultiplexKitIncludesSNaPshotMultiplexReadyReactionMix,
controlprimermix,andcontroltemplate.
SNaPshotPrimerFocusKitDesignedtodeterminetheapproximate
fragmentsizesgeneratedbyvariousprimersbeforeSNPgenotyping(criticalif
twooligonucleotidesproduceoverlappingsignalswhenrunsimultaneously)and
enablesthesettingoftightlociwindowsinGeneMapperSoftware.
GeneScan120LIZSizeStandardFivedyesizestandardthatisdesignedfor
reproduciblesizingofsmallfragmentanalysisdatageneratedwiththe
SNaPshotMultiplexSystems.Itaccuratelysizessamplesrangingfrom20to
120 nucleotides(nt).WhenusedwithGeneMapperSoftware,theGeneScan
120LIZSizeStandardeliminatestheneedformanualgenotyping.
MatrixStandardSetDS02Usedforspectralcalibration.
GeneMapperSoftwareGenotypeanalysisfordatageneratedwithSNaPshot
MultiplexSystems.
120

Additionally,theSNaPshotPrimerFocusKitallowsrapidassessmentofpotential
SNPoligonucleotides.Youcanpreviewallpotentialsinglebaseextensionproducts
andcalculatethemobilityrateforeachallele.Afterassessingthisdata,youcan
determinetheoptimalcombinationofSNPmarkersformultiplexing.Afteryou
determinethemultiplexformat,youcanusethereferencedatacreatedwiththePrimer
FocusKittoestablishmarkersandbinsetsintheGeneMapperSoftware,andreduce
thetimerequiredtodefineandeditbinsmanually.
Principle of the
analysis
Inthesinglebaseextensiontechnique,aunlabeledprimerisdesignedtoannealtothe
sequenceadjacenttotheSNPsite.Aftertheprimeranneals,thesinglebaseextension
occursbytheadditionofthecomplementarydyelabeledddNTP(dyeterminator)to
theannealedprimer.EachofthefourddNTPsisfluorescentlylabeledwithadifferent
colordye(Figure31).
Figure31 Single-base extension with dye-labeled ddNTPs
Unlabeled primer
Dye-labeled
ddNTPs
Template DNA
TheadditionofddNTPsyieldsmarkerfragmentsforthedifferentSNPallelesthatare
allthesamelength,butvarybycolor.
Afterelectrophoresisandfluorescencedetection,theallelesofasinglemarkerappear
asdifferentcoloredpeaksatroughlythesamesizeintheelectropherogramplot.The
sizeofthedifferentallelepeakswillvaryslightlyduetodifferencesinmolecular
weightofthedyes.
Advantages
Usesunlabeleduserdefinedprimersthatarecustomizedforyourtarget
Offersmultiplexingcapability(upto10plex,regardlessoftheirpositionsonthe
chromosomeortheamountofseparationfromneighboringSNPloci)
Sensitiveallelefrequencydetection(5%)
CompatiblewithallThermoFisherScientificgeneticanalyzers
AutomatedanalysisusingspecificGeneMapperSoftwaredataanalysismodule
Additionally,theSNaPshotkitcanbeusedforavarietyofotherapplications:
BACfingerprinting
DNAmethylation
Applications
(SNaPshot)
Lowtomediumthroughputlinkageandassociationstudies
Singlelocusfragmentanalysis
121

ScreenandconfirmSNPs
Screenforpriongenemutation
Screen and confirm SNPs

TheSNaPshotMultiplexSystemincludesavarietyofSNaPshotMultiplexKitsused
forSNPscreeningandvalidation.Eachkitoffersaonetubesinglebaseextension/
terminationreagenttolabelDNAfragments.
Screen for Prion gene mutations

ThesinglebasepairsensitivityoftheSNaPshotMultiplexSystemenablesyouto
accuratelyscreensamplesforcodondifferencesinpriongenes.Priondiseasesare
causedbyabnormallyfoldedisoformsofhostencodedproteins.UsetheSNaPshot
MultiplexSystemtoscreenforSNPsinthegenesthatcodefortheseproteins.For
instance,polymorphismsatcodons136,154and171ofthePrPgeneinsheepandgoats
canleadtoabnormallyfoldedisoformsoftheproteinproducttoresultinscrapie.
Instrument and
consumable
recommendations
Thermalcycler:Veriti,orGeneAmp 9700(forfastthermalcyclers,usea1C/
secondramprate),2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,or310instruments
Polymerandcapillaryarray:seeRunmodulesonpage69forthepolymerand
capillaryarraylengthcombinationssupportedoneachinstrument
GeneScan120LIZSizeStandard
DS02dyeset
IMPORTANT! Throughoutasetofexperiments,useallthesameequipment,run
parameters,polymers,dyes,andsoon.Consistentconditionsarerequiredto
avoidmobilityshiftsthatinterferewithaccurateinterpretationofdata.
122
Experiment and
primer design
recommendations
Minimumprimerlengthis23nt,howeveritisstronglyrecommendedthat
primersshorterthan36ntbetestedbeforemultiplexing.
HPLCpurificationofprimerslongerthan30ntisrecommended.Heterogeneous
primerpopulationswillleadtomajoranalysisissues.
Eachprimershouldhave23ntcomplimentarytothegDNAsequence.
Use5tailstocreatedifferentlengthprimers.
Addpoly(dGACT)togenerateasizedifferenceofatleast4to6nt.
Primerscanbecomplimentarytothe()strandoftheDNAifthe(+)strandis
difficulttoassay.
Alwaysrunanegativecontrol(notemplateDNA)whenevaluatinganewprimer.
Workflow
1. Designprimers.
2. PreparetemplatebyPCRoftarget,thencleanup
3. PrepareSNaPshotreactions
4. PostextensionbyPCR,thencleanup
6. Analyzedata
Data analysis
TheGeneMapperSoftwareincludesaSNaPshotDefaultanalysismethodthatyou
canuseasastartingpointforanalysis.
For more
information
Fordocumentsandpublications,seeSNPapplicationsonpage201.
AnextensivelistofpublicationsdemonstratingtheutilityoftheSNaPshotMultiplex
Systemisavailableatwww.lifetechnologies.com.
Fororderinginformation,seeSNaPshotKitsonpage198.
123
124

Fingerprinting
Overview........................................................... 125
Amplifiedfragmentlengthpolymorphism(AFLP)Analysis.............. 126
Terminalrestrictionfragmentlengthpolymorphism(TRFLP) ............. 131
BacterialArtificialChromosome(BAC)fingerprinting .................... 132
Highcoverageexpressionprofiling(HiCEP) ............................. 136
Intersimplesequencerepeat(ISSR)PCR ................................ 137
Overview
DNAfingerprintingisatechniquethatisusedtoidentifypatternsthatoccuringenetic
markers.Thesefingerprintsarespecifictoparticularorganisms.Anumberof
techniquesareavailableforfingerprinting.
125
Chapter8 Fingerprinting
Amplified fragment length polymorphism (AFLP) Analysis

Amplifiedfragmentlengthpolymorphism(AFLP)isamappingtechniqueusedto
visualizepolymorphismsingenomicDNA.TheAFLPsystemcombinesthe
restrictionfragmentlengthpolymorphism(RFLP)techniqueandpolymerasechain
reaction(PCR)togeneratealargenumberofamplifiedrestrictionfragmentsfrom
prepared,genomicDNA.Whenseparatedbyelectrophoresis,thesamplesyield
uniquebandpatternsthat,whenvisualizedbysouthernblotorfluorescencebased
fragmentanalysis,canbeusedforhighresolutiongenotyping,polymorphism
detection,orcladistics(Savelkouletal.1999).
Principle of the
analysis
TheAFLPprocedureinvolvesdigestinggenomicDNAtoproduceapopulationof
restrictionfragments,ligationofprimingsites,thenamplifiedbyPCR.(Goeletal.
2006.)ItissometimesconsideredavariationofrandomamplifiedpolymorphicDNA
(RAPD).
Figure32 AFLP analysis

Restriction
digestion
Ligation
Preselective
amplification
Selective
amplification
Electrophoresis
Sample 1
Sample 2
Sample 3
AFLPispossiblebecausetheabundantcomplexityineukaryoticgenomicDNA
meansthatitisstatisticallylikelythatenoughrestrictionfragmentswillbeshort
enoughtosuccessfullyproducePCRampliconsthatyieldauniquefingerprint
profile.
Advantages
126
ThepowerofAFLPanalysisderivesfromitsabilitytoquicklygeneratelarge
numbersofmarkerfragmentsforanyorganism,withoutpriorknowledgeofthe
genomicsequence.Inaddition,AFLPanalysisrequiresonlysmallamountsof
startingtemplateandcanbeusedforavarietyofgenomicDNAsamples.
Applications
Fingerprints,orAFLPbandpatterns,canbeusedformanypurposes.Forexample,
AFLPanalysisisoftenusedinplantresearchwherefingerprintscanbecomparedto
determinetheplantvarietyortocomparethesimilaritiesbetweendifferentplant
varieties.
ThermoFisherScientificprovideskitsforperformingAFLPonmicrobesandplants,
andreagentsthatareusefulforperformingAFLPonotherorganisms.Some
additionalapplicationsforAFLPanalysisinclude:
Moleculardiversitystudies(Zhaoetal.2006andJohnsonetal.2005.)
Phylogenystudies(Goeletal.2006.)
Breeding(Zhaoet.al.,ibid.)
Backcrossstudies(Johnsonetal.andGoeletal.,ibid.)
Mappingofclonedfragmentsinbacterialandyeastartificialchromosomes(BACs
andYACs)(Serra,etal.2006,Naimuddin,etal.2004)
Identifyingnewspeciesorsubspecies(Johnsonetal.ibid.,andSavelkoul,etal.
1999.)
TheAFLPkitsavailablefromThermoFisherScientificareoptimizedforplantsand
microbes.However,theyareanexcellentstartingpointforcustomAFLPexperiments
onotherorganisms(suchasfish).ContactyourThermoFisherScientificfield
applicationsspecialistformoreinformationonusingAppliedBiosystemsAFLPkits
toconductexperimentsinorganismsotherthanplantsormicrobes.
Instrument and
consumable
recommendations
Thermalcycler:Veriti(standardmodeonly),GeneAmp9700,2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,or310instruments
Polymer:seeRunmodulesonpage69forthepolymerandcapillaryarray
lengthcombinationssupportedoneachinstrument
Capillaryarray:
3500Seriesinstruments:50cm
3730Series,3130Series,and310instruments:36cm
GeneScan500ROXSizeStandard(includedinkits)
DS32MatrixStandard(DyeSetF)
Experiment and
primer design
recommendations
DNA extraction and purification

BecauseAFLPanalysisrequiresonlyasmallamountofDNA(50to500ng,ideally
10 to100ng),DNApurificationiscritical.
WerecommendthefollowingkitsforextractingDNAforAFLPanalysis:
PlantAnalysis:DNAzolReagentorPureLinkGenomicPlantDNAPurification
Kit
MicrobialAnalysis:PureLinkGenomicDNAMiniKit
127
Restriction
InAFLPexperimentsongenomesofunknowncontent,determinewhetherornot
yourgenomicDNArestrictsproperlywithEcoRIandMseIenzymes.
Ingeneral,theRegularPlantGenomeKitmodulesshouldproducequalitygenetic
fingerprintswithgenomesof5x108to6109basepairs,andtheSmallPlantGenome
Kitmoduleswithgenomesof5x107to5108basepairs.
EmpiricalguidelinessuggestthatiftheG+Ccontentofthegenomeis>65%,MseIwill
notgiveasignificantnumberoffragments.OptimalresultsareobtainedwithMseI
whentheG+Ccontentis<50%.EcoRIalsotendstoproducemorefragmentsin
G+Cpoorgenomes.IncaseswhereanorganismsG+Ccontentisunknown,the
effectivenessoftherestrictionenzymesmustbedeterminedempirically.
Primers
Fortheselectiveamplificationstep,theprimersthattargettheEcoRI/Abindingsiteare
fluorescentlylabeledatthe5end.TheprimersthattargettheMseI/Cbindingsiteare
unlabeled.
Youmayneedtooptimizetoidentifyprimercombinationsthatgeneratesufficient
uniquemarkerfragmentsforastudy.
Forexample,thePlantMappingKitscontaineightselectiveforwardprimersandeight
reverseprimerslabeledwiththe5FAM,NED,andJOEdyelabeled
fluorophores(DyeSetF).Thepossiblecombinationsofforwardandreverseprimers
provides128possibleprimercombinationsthathavebeentestedacrossseveralcrop
genomes,facilitatingidentificationoftheoptimalpair(s)foragivenorganismwithout
havingtodesign,synthesize,orperformqualitycontroltestsofcustomprimers.
TheAppendixoftheAFLPPlantMappingProtocol(Pub.no.4303146)showsprimer
combinationsthathavebeensuccessfullyusedforavarietyofplantspeciesandthe
AFLPMicrobialFingerprintingProtocol(Pub.no.402977)showsprimercombinations
thathavebeensuccessfullyusedforavarietyofmicrobialorganisms.(Notethatif
yourorganismofinterestdoesnotappearinthelist,youcanstillconductexperiments
bychoosingprimersfromthemostcloselyrelatedspeciesthatisavailable.)
Ingeneral,thestrategywithAFLPanalysisistogenerateinformativefragments,or
enoughfragmentssothatindividualsaredistinguishable.However,toomany
fragmentscomplicatetheanalysis,soyoumustempiricallydeterminetheoptimum
numberoffragmentsneededforadequatediscrimination.Asageneralrule,itisbest
tohavebetween50and200peaksasthefingerprintafteramplification.
Workflow
1. Restrictiondigestion
2. Ligation
3. Preselectiveamplification
4. Selectiveamplification
6. Dataanalysis
128
Data analysis
TheGeneMapperSoftwareincludesanAFLPDefaultanalysismethodthatyoucan
useasastartingpointforanalysis.
Thismethodcontainsanalysisparametersforpatternrecognitionoffragmentsacross
samplestogenerateafingerprintforeverysample.Thismethodcanbeusedto
analyzeanytypeofdatafromfragmentlengthpolymorphismassayssuchasAFLPor
TRFLP.Featuresofthesoftwareusefulforanalysisinclude:
Abilitytogenerateapanel(thecollectionofmarkers)fromsamplefilesthathave
beenaddedtoaproject.
SizingQualityandGenotypingQualityvaluesflagpoorqualitysamplesenabling
easyidentificationanddecreasemanualreview.
Automaticgenerationoffinalmarkergenotypesinastandardbinaryformat
where1representsthepresenceofagivenfragmentwhile0representsthe
absenceofthecorrespondingfragment.
Uptofourprofilesareexpectedforeachsamplebecause:
BoththeforwardandreversePCRprimersmaybefluorescentlylabeled
Tworestrictionenzymesareused
GeneratepanelsandbinsetsusingtheAFLPDefaultanalysismethod.Youcanthen
routinelyanalyzedatausingthispanel.
Achangeinthefragmentprofilecanbeindicatedbytheabsenceofapeakaswellasa
reductionintheheightofapeakwhencomparingdifferentsamples.
ThefollowingtwofiguresareexamplesoftypicalandpolymorphicAFLPreactions.
Figure33 Typical electropherogram of an AFLP reaction
129
Figure34 Polymorphic AFLP peaks
Thesepeakpatternsareautomaticallyconvertedtoatableofbinarymarkergenotypes
(Figure35),whichcanbeexportedandanalyzedforsimilarityandgenerationof
dendrogramsusingastatisticalsoftwarepackageorotherdownstreamanalysis
softwareforthistypeofclusteringanalysis.
Figure35 AFLP genotypes in GeneMapper Software
For more
information
130
Fordocumentsandpublications,seeAFLPapplicationsonpage200.
Fororderinginformation,seeOrderingInformationonpage193.
Terminal restriction fragment length polymorphism (T-RFLP)
Terminal restriction fragment length polymorphism (T-RFLP)

Overview
Terminalrestrictionfragmentlengthpolymorphism(TRFLP)analysisisamapping
techniqueusedtostudycomplexmicrobialcommunitiesbasedonvariationinthe
16S rRNAgene(OsbornandMooreet.al.).Itiscultureindependent,rapid,sensitive,
andreproducibleanddoesnotrequiregenomicsequenceinformation.
Principle of the
analysis
InTRFLPanalysis,fluorescentlylabeledDNAisdigestedwithrestrictionenzymes
thathave4basepairrecognitionsites.Thisstepgeneratesfluorescentlylabeled
terminalrestrictionfragments.Thefragmentsinthedigestarethenseparatedby
capillaryelectrophoresis.Profilescanthenbecomparedbetweensamples,ormatched
toadatabaseofknownspecies.
Applications
Examinemicrobialcommunitystructureandcommunitydynamicsinresponseto
changesindifferentenvironmentalparametersortostudybacterialpopulations
innaturalhabitats.
Studyofcomplexmicrobialcommunitiesindiverseenvironmentssuchassoil
(DerakshaniandLukowet.al.),marineandactivatedsludgesystems
(EschenhagenandSchuppleretal.)
Characterizeoralbacterialflorainsalivainhealthysubjectsversuspatientswith
periodontitis(SakamotoandTakeuchietal.).
PreliminaryscreeningofmicroorganismsbeforeanalysisusingApplied
BiosystemsMicroSEQMicrobialidentificationkits.
Instrument and
consumable
recommendations
ThisisaThermoFisherScientificdemonstratedprotocol.
Thermalcycler:Veriti,GeneAmp9700,2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,and310instruments
DS33MatrixStandard(DyeSetG)
131
Bacterial Artificial Chromosome (BAC) fingerprinting
Experiment and
primer design
recommendations
FollowtherecommendationsforAFLPanalysis.SeeExperimentandprimerdesign
recommendationsonpage127.
Workflow
DNA
extraction
PCR with
labeled
primers
Digestion with
restriction
enzymes
Data
analysis
Electrophoresis
1. DNAisolationandpurification.
2. PCRamplificationandrestrictionenzymedigestion.
3. Separationanddetectionofthedigestedproductsviaelectrophoresis.
4. Analysisofdatatogeneratethefragmentprofileforeachsample.
5. Clusteringanalysisbasedontheprofileofsamplesfromstep4.
Data analysis
TRFLPanalysisusesthesamedataanalysistechniqueasAFLP.SeeDataanalysison
page129.
For more
information
Fordocumentsandpublications,seeAFLPapplicationsonpage200.

Overview
BACfingerprintingprovidesanefficientandcosteffectivemethodofcharacterizing
largegenomicfragmentlibrariesforgenomesequencing,positionalcloning,and
physicalmappingefforts.RestrictionendonucleasedigestionofBACclonesfollowed
byfluorescentdyelabelingcanbeusedtogenerateaprofileorfingerprint.Overlap
betweenfingerprintsaresubsequentlyusedtoassemblecontiguoussequences
(contigs)intheconstructionofwholegenomephysicalmaps.Physicalmapsare
importantresourcesforgenomesequencingefforts,positionalcloning,comparative
genomics,andtodeterminethesizeandstructureofgenomes.
TheSNaPshotMultiplexKit(Luoetal.)providesaneffective,easy,andcosteffective
solutionforhighthroughputBACfingerprinting.
132
Principle of the
analysis
InBACfingerprintinganalysisusingtheSNaPshotMultiplexKit,BACclonesare
subjectedtorestrictionendonucleasetogeneratefragmentsofvariouslengthsthatend
inA,C,G,orT.TheSNaPshotchemistrythenlabelsthefragmentswiththe
correspondingbasesbysinglebaseextensiontocreateadistinctDNAfragment
patternorfingerprintforeachclone.Theclonesarethenmappedbasedontheorder
oftheoverlappingpartsoffingerprintswithotherclonesofthesamegenome.
Figure36 SNaPshot restriction fragment labeling
Recessed strand
Label template
SNaPshot
reaction mix
Color = genotype
Electrophoresis
Table19 Example of possible six-base cutters for restriction endonucleases and dyes used in
the SNaPshot Multiplex Kit
Restriction
endonuclease
ddNTP
Fluorescent
dye
Restriction fragment
color
EcoRI
GAATTC
dR6G
Green
BamHI
GGATCC
dR110
Blue
Yellow
XbaI
TCTAGA
dTAMRA
XhoI
CTCGAG
dROX
Red
None
HaeIII
Applications
Restriction
site
GGCC
WithBACfingerprinting,youcancreatewholegenomephysicalmapsthatare
importantresourcesfor:
Genomesequencing
Positionalcloning
Comparativegenomics
133
Instrument and
consumable
recommendations
ThisisaThermoFisherScientificdemonstratedprotocol.
IMPORTANT! BACfingerprintingisbaseduponpatternrecognition;therefore,data
analysisisfocusedonrelativesizeanddistribution.Werecommendusingadedicated
instrumentplatformtominimizelowrandomerrorcausedbysizingimprecision.
Thermalcycler:Veriti,GeneAmp 9700,2720
Geneticanalyzer:3500 Series,3730 Series,3130Series,and310instruments
Capillaryarray:
3500Seriesinstruments:50cm
3730Series,3130Series,and310instruments:36cm
DS33MatrixStandard(DyeSetG5)
Experiment and
primer design
recommendations
134
Protocolsmaydifferbasedonthekindofrestrictionendonucleasesandthe
BAC DNApurificationkitsthatareused.
EnzymaticdigestionandSNaPshotreagentlabelingcanbeperformedinone
tubeorinseparatereactions.
Workflow
1. Selectivebacterialgrowthofsinglecolonies.
2. BACpurificationbyrestrictionendonucleasedigestion.
3. RestrictionendonucleasedigestionoftheBACcloneswithseveraldifferent
enzymes.
4. SNaPshotreagentlabelingoffragments.Thedyelabeledprimersareboundto
theBACfragmentsbasedontheoverhangsleftbytherestrictionenzymes(see
Table19onpage133).
5. Postextensioncleanupoftheclones(notshownindiagram).
6. Capillaryelectrophoresis.
7. Dataanalysis.
8. Contigconstruction.
Data analysis
SeeBACapplicationsonpage200.
135
High coverage expression profiling (HiCEP)
For more
information
Fordocumentsandpublications,seeBACapplicationsonpage200.
High coverage expression profiling (HiCEP)

Overview
Thehighcoverageexpressionprofiling(HiCEP)methodoffragmentanalysiswas
developedtoaddresstheshortcomingsingeneexpressionprofilingandtoprovidea
sensitivemethodfordetectingalargeproportionoftranscriptsinbothknownand
unknowngenes,withalowfalsepositiverate.
AsanAFLPbasedgeneexpressionprofilingmethod,theHiCEPmethoddoesnot
requiresequenceinformationandhasareducedrateoffalsepositiveswithahigh
degreeofdetectionofbothcodingandnoncodingtranscripts.AfterHiCEPanalysis,
fragmentsofinterestcanbepurifiedandclonedfromagarosegelsandsequencedto
identifythetranscripts.Ifwholegenomesequenceinformationfortheorganismunder
studyisknown,thefragmentsofinterestcanbeidentifiedbybioinformaticprediction
usingthesequenceinformationavailablefrompublicdatabasesandtherestriction
enzymerecognitionsitesusedintheHiCEPworkflow.
Principle of the
analysis
HiCEPisanAFLPbasedmethod.TheanalysisinvolvesdigestinggenomicDNAto
produceapopulationofrestrictionfragments.Primingsitesarethenligatedontothe
endsoftherestrictionfragmentssothattheycanbeamplifiedbyPCR(Goeletal.
2006).
Applications
Fingerprinting
Recommendations
ThisisaCustomerdemonstratedprotocol.Forinformation,refertoHiCEP
applicationsonpage200.
Workflow
1. Synthesis
2. Digestion
3. Adaptorligationandpurification
4. Digestion
5. Adaptorligation
6. SelectivePCR
7. PostPCRpreparation
9. Dataanalysis
136
Inter-simple sequence repeat (ISSR) PCR
Figure37 HiCEP workflow
For more
information
Fordocumentsandpublications,seeHiCEPapplicationsonpage200.

Overview
Intersimplesequencerepeat(ISSR)PCRisafastandinexpensivegenotyping
techniquewithawiderangeofuses,includingthecharacterizationofgenetic
relatednessamongpopulations.ISSRPCRisagenotypingtechniquebasedon
variationfoundintheregionsbetweenmicrosatellites.
Inadditiontotheuseoflongfragmentsforaccurateanalysis,thistechniqueprovides
additionalbenefitsoveragarosegels.TheincreasedsensitivityofThermoFisher
Scientificgeneticanalyzersovertraditionalanalysismethodsroutinelyallowsthe
detectionofanorderofmagnitudemorepeaks,andthisincreasedresolutionresultsin
betterdiscriminationbetweenindividualsbeingcomparedinthepopulations.
However,theprimersthataredesignedtoannealtothediortrinucleotiderepeatscan
lackspecificityinPCRandareamajorcontributortoalackofreproducibility.Also,the
lackofcomplexityoftheISSRprimerscanleadtononspecificamplification,
particularlyifcoupledtopoorqualitygDNAextractionmethodsandsuboptimalPCR
amplificationconditions.
Principle of the
analysis
ISSRPCRusesasinglefluorescentlylabeledprimertotargettheregionbetween
identicalmicrosatellites(Figure38).AnISSRPCRprimercomprisesthreeparts:
Afluorescenttag
Eightdinucleotiderepeatunits(or6trinucleotiderepeatunits)
Oneormoreanchornucleotidesdesignedwithadualpurpose:totargettheend
ofamicrosatelliteregionandtopreventprimerdimerization.Morethan
100 primershavebeendevelopedforuseinISSRtechniques(UBCPrimerSet9,
2006 catalog).
137
Figure38 Example inter-simple sequence repeat (ISSR) region
BecauseISSRsaredominantmarkers,theamplifiedregionsinanISSRPCRarescored
asdiallelic.Betweenindividualswithinapopulation,changesintheamplified
productscanarisethroughstructuralchangestotheregion(insertionsordeletions)or
thelossofprimerbindingsites.
Advantages
Fasterandrequiresalowerstartupinvestmentthanothergenotyping
methodologiessuchasAFLPandRFLP.
SeveralstudieshavecomparedAFLPandISSRresultsandhavefoundISSR
preferablebecauseofthereducednumberofprotocolstepsrequiredandthe
smalleramountsofDNAconsumed.
Lessexpensiveandlesstimeconsumingthanmicrosatellitebasedgenotyping.
Noneedtocloneandcharacterizemicrosatellites.
Capillaryelectrophoresisdeliverssignificantlyhigherresolutionthantraditional
agarosegelelectrophoresis,thusincreasingtheamountofinformationobtained
fromeachexperiment.
Applications
ISSRhasbeenusedtoinvestigatemanyplantandanimalspeciesinthefollowing
techniques:
Geneticfingerprinting(BlairandPanaudet.al.1999)
Genetagging(AmmirajuandDholakiaet.al.2001)
Detectionofclonalvariation(LeroyandLeon2000)
Cultivaridentification(WangandWuet.al.2009)
Phylogeneticanalysis(GuptaandSouframanienet.al.2008)
Detectionofgenomicinstability(AndersonandBrenneret.al.2001)
Assessmentofhybridization(WolfeandXianget.al.1998)
TheversatilityofthisgenotypingtechniquemakesISSRusefulforresearchers
interestedindiversefieldssuchasconservationbiologyandcancerresearch.
Recommendations
138
ThisisaCustomerdemonstratedprotocol.Forinformation,refertoISSR
applicationsonpage200.
Experiment and
primer design
considerations
Cetyltrimethylammoniumbromide(CTAB)gDNAisolationdelivershighand
consistentamplification(DoyleandDoyle,1993).
InDNAamplification,primersandPCRmastermixesshouldbetestedfor
robustnessandconsistencywhenamplifyingISSRtargetsinbothspecies.
Subsequentlythermalcyclingconditionscanberefined,withparticularfocuson
primerannealingtemperatureandprimerannealingtime.
Foradditionalinformationonoptimization,refertoISSRGenotypingofEndangered
PlantsUsinganOptimizedWorkflowonpage200.
Workflow
Data analysis
IntheGeneMapperSoftware:
InthePanelManager,createapanelforeachdyecolor(primer)withbins,
centeredatwholebasepairs,onebasepairwidecoveringtheentirerangeof80to
1200bp(Figure39).
IntheGeneMapperManager,modifytheAFLPAnalysisMethod(Figure40).
Thismethoddetectspeaksaboveaminimumpeakheightasanalleleandapplies
abinarylabelofeither1or0forthepresenceofapeakinaparticularbin.
Figure39 Creating multiple ISSR bins and example of multiple bins centered at whole base pairs for the blue marker in an
ISSR Panel
139
Figure40 Example of the Allele tab settings for an ISSR analysis method
Example analysis
ISSRwasusedtocomparetwosamples:AgaveshawiishawiifromRosaritoandAgave
shawiishawiifromBorder.
Thefigurebelowshowsthedistinctpeakpatternsofthesetwoindividuals.
Figure41 Example ISSR data
Agave shawii shawii

from Rosarito
Agave shawii shawii

from Border
AfteranalyzingthedataintheGeneMapperSoftware,genotypeswereexportedand
evaluatedusingaspreadsheetprogramto:
AssesstheconsistencyofgenotypingforfourreplicateISSRPCRreactionsfor
eachprimeranalyzed.
Calculatetheallelessharedbetweenthereplicates.
Onlythosealleleswith100%concordancewerescoredastrueallelesandusedin
subsequentphylogeneticanalyses.
Truealleledataforeachindividualforeachprimerwereconcatenatedintoasinglelist
ofbinarystates.Thebinarydatawerethenanalyzedusingthephylogeneticsoftware
MrBayes(HuelsenbeckandRonquist;RonquistandHuelsenbeck).
140
ThephylogramsgeneratedfromtheMrBayessoftwareisshownbelow.Itindicates
withhighconfidencethatthreedistinctpopulationsofAgaveshawiishawii(alsoknown
asShawsAgave)existed.
Figure42 Phylogram generated using MrBayes software shows three distinct populations of
Agave. Individuals collected from Rosarito, Arroyo Honda, and Border are shown in gold, grey,
and purple, respectively. Highlighted individuals correspond to the data presented in Figure41
on page 140. Nodes in phylogram with posterior probability values above 95% are considered
to be informative in Monte Carlo Markov Chain (MCMC) Bayesian analysis (MrBayes).
For more
information
Fordocumentsandpublications,seeISSRapplicationsonpage200.
141
142
Relative Fluorescence Quantitation

(RFQ)
Overview........................................................... 143
Experimentandprimerdesignrecommendations........................ 144
LOHworkflow ...................................................... 145
Dataanalysis ........................................................ 145
Formoreinformation................................................. 146
MicrosatelliteInstability(MSI)andReplicationError(RER)................ 146
Overview
Relativefluorescencequantitation(RFQ)isatechniqueusedinavarietyoffragment
analysisapplicationstocomparepeakheightsacrosssamples.
Relativefluorescenceapplicationscomparepeakheightorareabetweentwosamples.
Commontechniquesinclude:
QualitativeFluorescence(QF)PCR
QuantitativeMultiplexPCRofShortFluorescentFragments(QMPSF)
MultiplexLigationdependentProbeAmplification(MLPA)
Principle of the
analysis
ThedataforanRFQexperimentcanbeobtainedwithmicrosatelliteorAFLP
analysis.
Peakheightorpeakareacanbeusedtocomparedifferencesinthesamemarkeracross
multiplesamples.However,youmayseeadifferenceinresultsdependingonwhether
peakheightorpeakareaisused.
IMPORTANT! VariationsinsignalintensityadverselyaffectsresultsinRFQ
experiments.Forinformationonminimizingvariation,seeOptimizingsignal
intensityonpage77.
AsanexamplemicrosatelliteRFQexperiment,thefigurebelowshowsan
electropherogramofamicrosatellitemarkerinDNAfromahealthyandtumor
sample.Thereducedpeakheightinthetumorsampleindicatespotentiallossof
heterozygosity(LOH)inthesample.
143
Chapter9 Relative Fluorescence Quantitation (RFQ)

Figure43 Example electropherogram of healthy and tumor samples
Applications
Screeningforlossofheterozygosity(LOH)usingmicrosatellitesorSingle
NucleotidePolymorphisms(SNPs)
Aneuploidyassays
Detectionoflargechromosomaldeletions
Multiplexligationdependantprobeamplification(MLPA)

Recommendations
Donotuseinternallylabeled([F]dNTPlabeled)fragmentsinquantitative
experiments.Variationsintheperfragmentnumberoflabelednucleotidesand
theincreasedpeakspreadingwiththismethodmakerelativequantitation
unreliable.
Formoreinformation,seeMicrosatelliteAnalysisonpage107,andAmplified
fragmentlengthpolymorphism(AFLP)Analysisonpage126.
Minimizing signal
intensity variation
Tominimizevariations,considertheionicstrengthofsamplesandconsumables.The
amountofDNAinjectedisinverselyproportionaltotheionicstrengthofthesolution:
Sampleshighinsaltresultinpoorinjections.PCRreactionsvaryinefficiency,
thereforesomereactionsmayresultinhigherionicconcentrationpost
amplification.
Conductivityofthesolventusedforinjectionwillaffectthesampleinjectionand
cancausevariationinpeakheight.
144
LOH workflow
Formoreinformation,see:
Optimizingsignalintensityonpage77
Irregularsignalintensitytroubleshootingonpage164
Relativefluorescencequantitationapplicationsonpage200
IMPORTANT! Preferentialamplificationcandecreasetheaccuracyofrelative
quantitationmeasurements.Formoreinformation,seePreferentialamplificationon
page31.
LOH workflow
1. Designtheprimersandselecttheprimerdyeset.
2. PCR:
RuntwoDNAsamplesfromeachindividual,forexample:
Onefromnormaltissue(N)
Onefromtumortissue(T)
Note: Somenormaltissuecontaminatingthetumortissuesampleistypical.
Run3to4independentinjectionsforeachsample(NandT)toobtain
sufficientlyaccuratequantitativeestimatesforsubsequentdataanalysis.
RuncontrolDNA:
AmplifyatleastonecontrolDNAsampleforeveryroundofPCR
amplification.
RunatleastoneinjectionofamplifiedcontrolDNAforeverysetof
microsatellitemarkersused.
RunatleastoneinjectionofamplifiedcontrolDNAwheneveryou
changethecapillaryorelectrophoresisconditions.
3. Capillaryelectrophoresis.
4. Dataanalysis.
Data analysis
Precise peak
detection
Optimizepeakdetectionparameterstoensureprecisepeakdetection.Formore
information,seeDataAnalysiswithGeneMapperSoftwareandPeakScanner
Softwareonpage89.
Ifnoisepeaksaredetected,increasetheMinimumPeakHalfWidthoruseastronger
smoothingoptionwhenanalyzingnoisydata.
Determining
relative quantities
Youcandeterminetherelativequantitiesoftwo5endlabeledfragmentsby
comparingthecorrespondingpeakareasorpeakheightsonaGeneMapperSoftware
orPeakScannerSoftwareelectropherogram.
145

Assessthereproducibilityofpeakheightandareaforeachnewanalysis.Notethe
following:
Useareaforslowmigratingorwidepeaksathighconcentration.
Useheightforsharppeaksatlowconcentration.
Thereisalinearrelationshipbetweenthemigrationtimeandthereproducibility.
Asthemigrationtimeincreases,thepeakwidthandareaincrease.Therefore,fast
migratingpeaksresultinhigherreproducibilityasmeasuredbythepeakarea.
However,improvedreproducibilitycalculatedusingpeakheighthasbeen
observedasthemigrationtimeincreases(ShihabiandHinsdale,1995).
Determining
relative number of
molecules
Todeterminetherelativenumberofmoleculesoftwodifferentsizedfragments,
calculatetheratioofrespectivepeakareasorheights.Makesuretocomparepeakarea
topeakareaorpeakheighttopeakheight:
Iftwofragmentsaresimilarinsize,comparepeakheights,especiallyifthepeaks
overlapslightly.Ifthepeaksarewelldefined,peakareaandpeakheightwillgive
similarresults.Ifthepeaksareirregularlyshapedorhaveshoulders,peakheights
willoftengivebetterresultsthanpeakareas.
Iftwofragmentsdiffergreatlyinsize,comparepeakareasbecauselargepeaks
tendtospreadconsiderablymorethansmallpeaks.

Fordocumentsandpublications,seeLOHapplicationsonpage200.
Microsatellite Instability (MSI) and Replication Error (RER)

Microsatelliteinstability(MSI)describesthereducedfidelityduringthereplicationof
repetitiveDNAoftenoccurringintumorcells.Itisthoughttobecausedbystrand
slippageduringDNAreplicationduetomutationsinDNAmismatchrepairgenes.
MSIleadstotheappearanceofmultipleallelesatmicrosatelliteloci.Replicationerror
(RER)isusuallydefinedasMSIatmultiplemicrosatellitemarkersorloci.The
appearanceofnumerousextraallelesatlowermolecularweightsinthetumorsample
(Figure44bottompanel)indicatessignificantgenomicinstability.
146
Figure44 Microsatellite instability identified by lower peak signal intensity in the tumor sample
Normal
Tumor
ThetechniquefordetectingRERinvolvescomparingmicrosatelliteallelesafterPCR
amplificationinnormalandtumorsamplesfromthesamehost.Youcalculatearaw
RERscoreusinganalgebraicformulathatquantifiestherelativestrengthofthe
stutterpeaksinthetwosamplesafternormalizingfordifferencesinPCRefficiency.
Whilebothmicrosatelliteinstabilityandlossofheterozygosityareindicativeof
canceroustissue,ifanelectropherogramshowsRERatagivenmarkerlocation,an
LOHcalculationforthatalleleregioniscomplicatedoreveninvalid(Canzianetal.
1996).WedonotrecommendLOHcalculationsinregionsthatshowclearsignsof
RER.
147
148

10
Additional Applications
DNAmethylation .................................................... 149
DNA methylation
Thestudyofmethylation/epigeneticsisemergingasanimportantcomponentof
cancerresearch.Inatypicalassaytodetectmethylation,bisulfitetreatmentofDNA
deaminatesnonmethylatedcytosineandconvertsittouracilwhilemethylated
cytosineremainsunreactive.ThesubsequentstepofPCRamplificationconvertsuracil
basestothymine.UsetheSNaPshotsystemtoquantitativelydetectthebase
differencesintreatedanduntreatedsamplestolearnthemethylationstatusofthe
samples.
Formoreinformation,seeMethylationapplicationsonpage200.
149
10
150
Chapter10 Additional Applications

DNA methylation
11
Troubleshooting
Troubleshootingworkflow............................................ 152
Refertothefollowingsectionsfortroubleshootingsolutionsandinformationonhow
eachcomponentofthesystemcanaffectdata:
Checkingdataquality ................................................ 153
Runningcontrolstoisolateaproblem................................... 156
Sampleissues ....................................................... 158
Reagentandconsumableissues ........................................ 159
Instrumentandambientconditionissues................................ 160
Refertothefollowingsectionsforsymptomtroubleshootinginformation:
Symptomsyoumayobserve ........................................... 162
Irregularsignalintensitytroubleshooting ............................... 164
Migrationtroubleshooting ............................................ 168
Abnormalpeakmorphologytroubleshooting ............................ 169
Extrapeakstroubleshooting ........................................... 172
PCRtroubleshooting ................................................. 176
Irregularbaselinetroubleshooting...................................... 178
Instrumentationtroubleshooting ....................................... 180
SizingorSizeQuality(SQ)troubleshooting.............................. 182
GeneMapperSoftwaretroubleshooting ................................ 187
Preamplificationgeltroubleshooting ................................... 190
Refertothefollowingsectionsforprocedurestosolveissues:
Desalting ........................................................... 190
Evaluating310GeneticAnalyzermulticomponentmatrixquality .......... 191
151
11
Chapter11 Troubleshooting
Troubleshooting workflow
Troubleshooting workflow
Problemswithdatacanbecausedduringanystepoftheexperiment.
DNA isolation
and purification
(template)
Sample reactions
(assay workflows
and kit protocols)
Sample plating
Instrument run
setup
Instrument run
Data collection
Data analysis
Interpretation
Whentroubleshooting,followthisworkflowtoidentifytheproblem.
1. Makesureyouunderstandthebasicsoftheexperiment:
Thechemistry
Labelingofthesamples
Howthegeneticanalyzercollectsdata
Howthedataanalysissoftwareperformssizingandpeakdetection
Reviewtheexperimentforerrorsinprimerdesign,samplequantitationand
purification,pipettingproblems,softwarepreferencesettingsandothercommon
mistakes.
2. Examinethedata.Evaluatetheproblemasspecificallyaspossible:
Isitaproblemwiththesamplepeaks,thebaseline,orthepeaksofonlyone
color?
Lookforpatterns:Doestheproblemexistinallpartsoftherunordoesit
affectonlyDNAfragmentsofacertainlength?inaspecificcapillary?ina
certainareaoftheplate?multipleruns?
Istheproblemvisibleinrawdata?analyzeddata?logfiles?
Continuetorefinethedescriptionoftheproblemasspecificallyandthoroughly
aspossible.
3. Ingeneral,checkfirstfortheissuesthatcanberesolvedmosteasily.Review:
Dataquality
Analysissettings
Datacollection
Experimentalsetup
Formoretroubleshootinginformation,seeyourinstrumentandsoftwareuserguides
andthedocumentslistedinDocumentationandSupportonpage 199.
152
Checking data quality
11

Sizing Quality (SQ)
PQV description
TheGeneMapperSoftwareSQPQVreflectsthesimilaritybetweenthefragment
patterndefinedbythesizestandarddefinitionandtheactualdistributionof
sizestandardpeaksinthesampledata.ThemetricoftheSizingQualitytestisa
combinationofseveralvalueswhichmeasurethesuccessofthealgorithmsthat:
Identifyandeliminateprimerpeaksbasedonpeakshape
Performsizematching(ratiomatching)
Makeasizecallingcurveusingthesizingmethodspecifiedintheanalysis
method.(formoreinformation,seeGeneMapperSoftwaresizingmethodson
page 100)
Checking samples
with
yellow and
red SQ samples
ReviewthedataofthesizestandardsthatfailtheSQPQVasdescribedbelow.For
moreinformation,seeSizingorSizeQuality(SQ)troubleshootingonpage 182.
1. IntheSamplestaboftheGeneMapperwindow,click
(AnalysisLow
QualitytoTop)tosortthedatasothatthesamplesthatproducederrorsappearat
thetopofthetable.
2. IntheSamplestab,selecttherowsforthesample(s)thatdisplay
(Check)or
(Fail)intheSQcolumn.
3. Click
(AnalysisSizeMatchEditor)toviewthesizinginformationforthe
selectedsample(s).
4. IntheNavigationPaneoftheSizeMatchEditor,selectasamplefiletodisplaythe
sizingdatafortheassociatedsample.
153
11
5. Reviewthedataforthefollowingqualities:
Signalstrength Thesignalstrength(peakheight)ofallpeaksmustexceed
thePeakAmplitudeThresholdsdefinedintheanalysismethodusedto
analyzethedata.
Correctsizecalls/labels Allpeakslistedinthesizestandarddefinition
mustbecorrectlyidentifiedbythesoftware.Thelabelsabovethepeaksmust
beinsequentialorderfromlefttoright,lowtohigh.
Evennessofsignalstrength Allpeaksshouldhaverelativelyuniform
signalstrengths.
Note: TomagnifytheplotoftheSizeMatchestab,dragthemousecursor(
acrossaregionofthexoryaxis.
Examining the raw

data for
red SQ
Examine the
sample info, raw
data, and EPT trace
Datafor
SQsamplesisdisplayedonlyintheRawDatatab(seeExaminethe
sampleinfo,rawdata,andEPTtracebelow).
1. IntheProjectwindow,selectasampleinthenavigationpane.
2. Checkforerrormessages.
Note: Ifthiserror
messageis
displayedatany
timewhenyou
areusingthe
software,check
theInfotabto
determinethe
error.
3. Reviewthesampleinformation.Ensurethatthecorrectanalysissettingsanddata
collectionsettingwereused.
154
11
4. ClicktheRawDatatab.
Note: TheRawDatatabistheonlyplaceinthesoftwareinwhichyoucanview:
Negativebaselines
Rundatafor
SQsamples
Theexamplebelowillustratesgoodqualityrawdataformultiplexed
microsatellitedata.
5. ClicktheEPTDatatab.Reviewthecurrent,voltage,temperature,andpower
throughouttheelectrophoresisrun.Largefluctuationsinthevaluescanresultin
poorqualitydata.
TheexamplebelowillustratesagoodqualityEPTtrace.Thevaluesforthetrace
maydifferdependingontherunmoduleused,buttheshapeofthetraceshould
besimilartotheexamplebelow.
155
11
Running controls to isolate a problem
Note: TracecolorsdifferbetweentheDataCollectionSoftwareandtheGeneMapper
Software.
Formoreinformation,seeInstrumentationtroubleshootingonpage 180

Tosimplifytroubleshooting,ThermoFisherScientificrecommendsthatyourun
controlswitheveryrunformulticapillaryinstrumentsoreachsetofrunson310
instruments.
Inadditiontocontrolsincludedineachrun,youcanrunsizestandards,installation
standards,agarosegels,orDNAtemplatecontrolswhenadditionaltroubleshootingis
required.
Size standard
1. Performarunwithonlysizestandard,usingoneofthedefaultrunmodulesthat
areprovidedwiththesoftware:
a. Vortexthesizestandardfor1minute.
b. Addtoeachwellofaplate:
3500 Series, 3730 Series, and 3130
Series instruments
0.5 uL of size standard
0.5 uL of size standard

9.5 uL of fresh
Hi-Di
310 instruments
Formamide
11.5 uL of fresh Hi-Di Formamide
c. Runtheplate.
2. Examinethepeakmorphology:
Ifthepeakmorphologychanges,forexample,peaksbecomebroader,are
tailing,orarebelow50RFU,thentheproblemmaybeintheinstrument,
reagents,orHiDiFormamide.
Ifthepeakprofilesforthesizestandardalonearesharpandverywell
defined,addyourproducttothesamewellsandrerun.
Ifthepeakmorphologythenchanges,forexamplepeaksbecomebroader,
showtailing,arelessthan50RFU,thencontaminationmaybecontributing
totheproblem.
Note: Thesizestandardpeakheightsareaffectedbythepresenceofsample
becausethesampleintroducessaltandcompetesforentryintothecapillary
duringinjection.
Ifthesizequalityfailsinthepresenceofsample,itindicatesaproblemwiththe
PCRproduct,forexample,itmaycontaintoomuchsalt.
Installation
standard
156
InstallationstandardscontainpooledPCRproductsamplifiedfrommicrosatelliteloci
presentinCEPHindividual1347.
11
Toruntheinstallationstandard:
Forallplatforms,youcanloadtheinstallationstandardasaregularrunandview
theresultsintheGeneMapperSoftware.Forinstructions,refertothe
appropriateinstrumentuserguide.
Alternatively,fora3500Seriesinstrument,youcanrunaperformancecheck
whichproducesareportquantifyingeachpeak(refertoyourinstrumentuser
guide).
ThermoFisherScientificcurrentlysuppliesthefollowinginstallationstandardsforits
capillaryelectrophoresisinstruments(seeInstallationstandardsonpage 197forpart
numbers):
If you use...
Then use...
Which uses these dyes:
GeneScan 600 LIZ Size

Standard v2.0
DS-33 GeneScan
Installation Standards
6-FAM, VIC, NED, and

PET dyes
GeneScan 500 LIZ Size

Standard
DS-33 GeneScan
6-FAM, VIC, NED, and

PET dyes
GeneScan 500 ROX
DS-30 GeneScan
6-FAM, HEX, and NED

dyes
Agarose gel
RunthePCRproductthroughanagarosegeliftheelectropherogramshows
miscellaneousunexpectedpeakswhichmaybeduetounincorporatedproduct.
Resultsfromthegelwillhelptodetermineifyoursampleiscontaminated.
DNA template
control
YoucanuseaDNAtemplatecontrol(forexample,CEPH134702ControlDNAuseful
forhumantargetprimers)asaprocesscontroltoensurethatsamplepreparation,PCR,
andelectrophoresisyieldtheexpectedresults.
Theresultscanhelpyoudeterminewhetherfailedreactionsarecausedbypoor
templatequality,problemswiththecontrol,orproblemswiththeprimers:
1. RunanagarosegeltoseparatethePCRproducts.
2. Runcontrolprimerwithcontroltemplatetoeliminatecontaminatedreagentsasa
possiblecause.
3. Runcontroltemplatewithyourprimerstoeliminateyourprimersasapossible
cause.
4. Runcontrolprimerswithtemplatetoeliminatetemplateasapossiblecause.
Possible cause if the control fails
Action
Incorrect PCR thermal cycling conditions
Choose correct temperature control

parameters (refer to your instrument user
guide).
Pipetting errors
Calibrate pipettes, attach tips firmly, and

check technique.
Combined reagents not spun to bottom of

tube
Place all reagents in bottom of tube. Spin

briefly after combining.
157
11
Sample issues
Possible cause if the control fails
Action
Combined reagents left at room temperature

or on ice for extended periods of time
Put tubes in block immediately after

reagents are combined.
Restriction digest incomplete (AFLP

applications)
Repeat the restriction-ligation.

Make sure that the volume of enzyme added
does not cause the amount of glycerol to be
>5%, which can lead to EcoRI* (star) activity.
Sample issues
Sample
concentration
Ifthesampleconcentrationistoolow,thesignaltonoiseratiomaybetoolowto
discriminatebetweensamplepeaksandbackgroundfluctuations.
IfthesampleDNAconcentrationistoohigh,signalintensitymaybeoffscaleor
saturatedandcancause:
Splitpeaks
Raisedbaseline
Pulluppeakswhichcanaffectsizingandaccuracyofgenotypes
Adjustsampleconcentrationtoensuresignalintensityiswithintherecommended
rangeforyourinstrument:
Instrument
Recommended signal
level
Fluorescence saturation
3500 Series
17510,000 RFU
30,000 RFU
3730 Series
15010,000 RFU
30,000 RFU
3130 Series
1504000 RFU
8000 RFU
310
1504000 RFU
8000 RFU
Ifnecessary,dilutePCRproducts(beforeincludingthesizestandardinthereagent
mix)sothatthefinalallelepeakheightfallsintotherecommendedrangeforthe
instrument.
Sample
contamination
Samplecontaminationcanmimicadegradedcapillary.Youcandetermineifthe
capillaryissueiscausedbysamplecontaminationbyrunningasizestandardand
formamideonly(seeRunningcontrolstoisolateaproblemonpage 156).
Salt concentration
SaltanionscompetewithnegativelychargedDNAforentryintothecapillaryduring
electrokineticinjection.Asthesaltconcentrationofasampleincreases,lessDNAwill
enterthecapillary,decreasingthefluorescencesignal.Excesssaltcanalsoprecipitate
theDNAinthesampletubeinthepresenceofformamide.
158
Reagent and consumable issues
11
Reagent and consumable issues

IMPORTANT! Foreverychemical,readtheSafetyDataSheets(SDSs)andfollowthe
Laboratory water
PoorqualitylaboratorywatersystemsandcleaningreagentscanadverselyaffectPCR
efficiency,sampleresolution,andsignalintensity.
PCR reagents
ExpiredPCRreagentscancausedecreasedDNAtemplateconcentration.
Hi-Di formamide
ImproperlystoredHiDiFormamidecancause:
Incompletedenaturationofboththesizestandardandsamplepeaks
AlteredpHoftheloadingsolution
Tailingpeaks
Artifacts
Decreasedsignal
EnsurethatyoudonotcontaminateHiDiFormamidewhensettingupsamples.
Formoreinformation,seeHiDiFormamidestorageonpage 82.
Polymer
Degradedorexpiredpolymer,orpolymerthatisleftatambienttemperaturefor
>7 days,cancause:
Reducedcapillaryarraylife(thenumberofrunsperarray)
Reducedresolutionduetoincreasedconductivity(oftencausedbythehydrolysis
ofureainthepolymer)
Lowcurrent
Artifactpeaksfromdegradedpolymer
Reducedsizingprecision:
Sizingdifferencesbetweenvarioustypesofpolymeraremoreapparentfor
sequences<50bp.
Fragments<50bprunon3730/3730xlinstrumentswithPOP7polymer
mayhaveslightlylowersizingprecision.
Polymerthatisleftatambienttemperatureforextendedperiodsoftimecancause
microbubblesinthepump.
Coldpolymercancausebubbles.
Ensurethatpolymerisatroomtemperature.Allowpolymertoequilibratetoroom
temperatureandpressure.Loosenthelidsealatleast30to60minutesbeforeuse.Do
notleavethelidoffthepolymerbottle,asdustmaycontaminatestock,causingspikes
indata.
Note: Donotshakepolymerorintroducebubbles.
159
11
Instrument and ambient condition issues
Size standard
Sizingqualityissuescanbecausedbyadegradedorimproperlystoredsizestandard.
AsizestandardcanbedegradedbyusingimproperlystoredHiDiFormamide
(seeHiDiFormamidestorageonpage 82).
Ionic buffer
strength
(not applicable to
3500 Series
instruments)
Conductivitychangesinthebufferaffecttheruncurrentandcancausethefollowing:
Decreasedsampleresolution
Slowerthanexpectedmigrationofsizestandardpeaks
Lowcurrent
Possiblecausesofbufferissues:
Waterimpurities
Highsaltconcentration
Expiredorincorrectlystoredbuffer

Capillary array
Degradedcapillaryarrayscancause:
Decreasedsampleresolution
Broad,laggingpeaks
Possiblecausesofdegradedcapillaryarrayperformance:
Capillaryarraylifeisexceeded
Capillaryarrayisleftidleordriesout
PoorqualityDNAorwaterordegradedHiDiformamidehasintroduced
contaminantsthatultimatelyaffectthecurrentflowthroughthecapillaries
Waterwashisnotperformedasrecommended,orcontaminatedwaterisusedfor
thewash
Cloggedcapillary
Ifthesamecapillaryalwaysfails,runHiDiFormamideblanks,thenan
installationstandardorsizestandardascontrolsthroughthecapillary(see
Runningcontrolstoisolateaproblemonpage 156).
Note: Samplecontaminationcanmimicadegradedcapillary.Youcandetermine
ifthecapillaryissueiscausedbysamplecontaminationbyrunningcleanDNA
samplesorthesizestandardaloneasacontrol.
Bubblesinthecapillaries
Arcing
Pump: large
bubbles
Largebubblescanaffectallormanyofthesamplesinarun.
Largebubblesinthepumporblockscanaffectthecurrentandcancausethe
following:
Nocurrentwhenvoltageisapplied(theflowofionsisblockedbythebubble)
Arunstopsduringinitializationiftheinstrumentdetectsunstablecurrent
Leakdetectederrormessageastheairbubblecompresseswhentheplunger
movesdowntofillthearray
160
11
Arcing
Earlylossofresolutiononcertaincapillaries
Iflargebubblesarepresent,youcanusuallyseethemintheupperpolymerblock,near
thearrayend.Refertotheinstrumentuserguideforinformationonremoving
bubbles.
Pump: small
bubbles
Smallmicrobubblesusuallyonlyaffectsinglecapillaries.
Microbubblesinthepolymerpathcancause:
Currentfluctuationsornocurrent
Decreasedresolution
Possiblecausesofsmallbubblesinthepump:
Polymeris:
Notatroomtemperature.Allowpolymertoequilibratetoroomtemperature
andpressure.Crackopenlidsealforatleast30to60minutesbeforeuse.Do
notleavethelidoffthepolymerbottle,asdustmaycontaminatestock,
causingspikesindata.
Newlyinstalled
Valves,syringes,orthearrayportarenotscrewedtightlyinplace
Refertotheinstrumentuserguideforinformationonremovingbubbles.
Pump: polymer
leaks
Polymerleakscancause:
Formationofcrystalswhichintroducecontaminantsthatcanaffecttheconditions
ofyourrun.Ifthepumpisnotadequatelypushingpolymerthroughthearray,the
arraycanclogorbecomecontaminated.
Lossofresolution
Autosampler
misalignment
Autosamplermisalignmentcancauseconsistentfailuresinthesamewellsorrowsofa
plate.
Temperature/
humidity
Drasticchangesinroomtemperatureandhumiditycancausedistinctchangesin
migration.
Matrix/spectral
Issues
Incorrectmatrix/calibration,orusingdifferentconditionstocalibratethanyoudoto
runsamplescancausethefollowing:
Raisedbaseline
Negativepeaks
Peaksunderpeaks(especiallyifthehighestpeakisnotoffscale)
Multipledyecolorsbeingdetectedasonedyecolor(hasbeenobservedwhen
running5dyesampleswitha4dyematrix)
161
11
Symptoms you may observe

Irregular signal
intensity
Symptom
No signal or low signal
164
Signal intensity is too high or oversaturated
166
Ski-slope peak pattern
166
Decreased signal
167
Size-standard signal and sample signal are not balanced
168
Migration issues
Symptom
See page
Size-standard peaks are not migrating as expected during

a normal run time
168
Sizing precision is low
168
Abnormal peak
morphology
Symptom
See page
Poor resolution
169
Loss of resolution
169
Broad, lagging peaks
170
Tailing peaks
170
Uneven peak heights in dyes in multiplexed sample
171
Sudden loss of signal in all samples
171
Multiple dye colors are detected as one dye color
171
Extra peaks
Symptom
See page
Extra peaks
172
Pull-up peaks
173
Data spikes
174
Split peaks
175
Many small extraneous peaks appearing next to a highintensity peak
175
PCR
162
See page
Symptom
See page
Poor priming resulting in weak signal
176
Amplified DNA concentration is lower than expected
176
PCR inhibition
176
Contamination with exogenous DNA
176
Poor amplification, nonspecific amplification
177
Symptom
See page
Poor amplification, nonspecific amplification in restrictionligation experiments (AFLP only)
177
Hairpin secondary structures in PCR primers
177
Primer/dimer formation
178
Irregular baseline
Symptom
See page
Constant elevated signal in raw data
178
Baseline waterfall
178
Noisy baseline
179
Adequate signal strength with noisy data
179
Instrumentation
Symptom
See page
Low or fluctuating current
180
Drop-off of current signal
181
Current too high
180
Instrument has stopped running and red light is on. There

are black marks inside the instrument.
181
Symptom
See page
No sample plot is displayed for a sample with the error No

sizing data
183
Size Match Editor does not display peak data
183
Missing size-standard peaks
183
Smaller size-standard peaks are not labeled
184
Larger size-standard peaks are not present in trace
184
Extra peaks in size-standard trace
185
Sizing failures occur in a regular pattern (the same wells

fail repeatedly)
186
Noise peaks are detected as size-standard peaks
186
Size call inaccurate for known DNA sample
186
Sizing or size
quality
GeneMapper
Software
Symptom
See page
GeneMapper Software error message
187
Error Message: The bin set in the analysis method does not
match the panel used for analysis.
187
al? label or alleles are not falling within bins
188
Allele not labeled
188
11
163
11
Irregular signal intensity troubleshooting
Symptom
See page
Data not sorted by name
188
When adding samples to a project, the expected data files

are not listed in the Add Samples to Project dialog box
188
Genotypes tab is grayed
189
Two peaks do not separate and are detected as one peak
189

Symptom
Possible cause
Action
Peak Amplitude Threshold
164
Poor or non-specific
amplification.
See PCR troubleshooting on page 176.
PCR inhibition.
Sample was prepared with

water instead of Hi-Di
Formamide.
Prepare the sample with Hi-Di Formamide.
Degraded or incorrectly stored

Hi-Di Formamide.
Use fresh, properly stored Hi-Di Formamide. See

Hi-Di Formamide storage on page 82.
Air bubble at bottom of sample

tube.
Centrifuge the plate before running.
See Non-specific amplification on page 32 for

more information.
Symptom
(continued)
Possible cause
High salt concentration.
11
Action
Salt preferentially injects smaller fragments and
inhibits injection of larger fragments, so the
majority of salt may have been injected in the first
injection.
Re-inject the sample.
If signal intensity does not increase, see
Desalting on page 190.
Injection time too low.
See Optimizing electrokinetic injection

parameters on page 78.
Sample concentration too low.
See Optimizing sample loading concentration on

page 76.
Sample volume too low.
Sample volume must be 10 L for 3500 Series,

3730 Series, and 3130 Series instruments.
Sample volume must be 12 L for 310
instruments.
Autosampler is misaligned.
3500 Series, 3730 Series, and 3130 Series

instruments: Fill wells with 0.5 L size standard
and 9.5 L sample, then re-inject. If the signal
is still missing, contact Thermo Fisher Scientific
310 instruments: Recalibrate autosampler.
165
11
Symptom
Signal intensity is too high or
saturated
Possible cause
Action
Raw data: pull-down peaks
Zoomed view of figure above
Sample concentration too high.
Decrease sample concentration.

Decrease injection time.
Ski slope peak pattern of

sample peaks but size standard
peak heights do not decrease
Sample concentration too high

in amplification step or
insufficient primer is present.
166
Optimize ratio of DNA template and primer.
Symptom
Decreased signal
11
Possible cause
Action
Degraded or improperly stored

Hi-Di Formamide.

Expired or incorrectly stored

reagents.
Use fresh reagents.
Degraded primers.
Store unused primers at 15 to 25C. Do not

expose fluorescent dye-labeled primers to light for
long periods of time.
Size-standard concentration is
too high.
Although the data is still sized properly, decrease

size-standard concentration to balance peaks in
future runs (see Size-standard peak intensity on
page 42).
Size-standard signal and

sample signal are not balanced
167
11
Migration troubleshooting
Migration troubleshooting
Symptom
Size-standard peaks are not
migrating as expected
during a normal run time
Possible Cause
Poor quality sample.
Degraded or frozen
polymer.
Action
Prepare fresh buffer (not applicable to 3500 Series
instruments).
Water used to dilute

buffe.r
Poor-quality formamide.
Fluctuations in ambient
temperature and/or
humidity.
Incorrect oven
temperature.
Old array or capillary.
Contaminants.
Low ionic buffer strength.
Sizing precision is low
Incorrect capillary length

(Length to Detector) or run
module was selected.
Specify correct capillary length or run module.
Variation in ambient
temperature causes faster
or slower migration rates.
Ensure ambient temperature is stable.
Analyzing small fragments

<50 bp.
Sizing differences between various types of polymer

are more apparent for sequences <50 bp.
Fragments <50 bp run on 3730/3730xl Series
instruments with POP- 7 polymer may have slightly
lower sizing precision than expected.
168
Abnormal peak morphology troubleshooting
11

Symptom
Poor resolution
Possible cause
Action
High sample concentration.
Dilute the sample before adding to formamide/

size-standard mix.
Injection time and/or voltage too

high.

Wrong capillary array and/or

polymer used.
Use appropriate capillary array or polymer for your

application.
Incomplete strand separation

due to insufficient heat
denaturation.
Make sure the samples are heated at 95C for

3 to 5 minutes, then immediately placed on ice for
2 to 3 minutes before loading.
Pump, polymer block, or septa

contaminated with chemicals
during cleaning.
1. Perform a water wash. Refer to the instrument

user guide for information.
Incomplete replacement of
polymer between runs.
1. Check the polymer delivery system for leaks,

looking for residue in and around the polymer
block area.
Loss of resolution
2. Replace polymer, buffer, septa and water/waste

with fresh materials.
2. Check the pin valve for signs of arcing on the tip.

Black markings within the block channel are
also a sign of an arcing event.
3. Check for polymer in the anode buffer jar. If you
see evidence of a leak, retighten connections,
then run the sample again.
Sample or reagent is
contaminated.
Use fresh samples and reagents.
169
11
Symptom
Loss of resolution (continued)
Possible cause
Action
injection.
Bubbles or debris in polymer

path.
Remove bubbles or clean the polymer path. Refer

to the instrument user guide for information.
Capillary array degrading.
1. Perform a water wash through the polymer

delivery system. Refer to the instrument user
guide for information.
2. Replace the capillary/array.
3. Run a size standard.
4. If the problem is present in the size standard,
replace reagents, then run your samples again.
Samples are degraded because

they have been sitting in the
instrument >24 hours.
Run samples as soon as possible after

preparation.
Expired or degraded polymer,

Hi-Di Formamide, buffer, or
water.
Replace the reagent, then run your samples again.

Instrument current problem.
See Instrumentation troubleshooting on

page 180.
Use of non-Thermo Fisher

Scientific reagents.
1. Perform a water wash on all components of the

system using the wizard in Data Collection
Software.
2. Replace reagents with Thermo Fisher Scientific
products.
Broad, lagging peaks
Old or clogged capillary array.
Replace the capillary array or flush the capillary

array with polymer.
Tailing peaks
Degraded or improperly stored

Hi-Di Formamide.

170
Symptom
Possible cause
11
Action
Uneven peak heights in dyes in

multiplexed sample
Sample preparation issues.
Optimize sample preparation and PCR.
Preferential amplification of
PCR products.
see PCR troubleshooting on page 176.
Selection of dyes is not optimal

(for example, a low-intensity
sample peak is labeled with a
low-intensity dye).
Select appropriate dye. For information, see

Points to consider when selecting dyes for custom
primers on page 39.
Concentration of some samples

is too high.
Adjust the pooling ratio before PCR (see

Multiplexing (pooling) strategies on page 27).
If overall concentration is too high, dilute pooled
samples with deionized water before PCR.
Increasing the MgCl2 concentration of some
samples can reduce the disparity in peak heights,
but may also increase the amplification of
non-specific products (background).
Sudden loss of signal in all

samples
Instrument laser power or

current problem.
Check laser power and current in the EPT window

(see Examine the sample info, raw data, and EPT
trace on page 154).
Multiple dye colors are detected

as one dye color
5-dye samples were run with a

4-dye matrix.
Repeat run with correct dye set.
171
11
Extra peaks troubleshooting

Symptom
Extra peaks
Possible cause
Action
Pull-up or cross-talk due to

saturated data in a dye color (for
example, a high intensity blue
peak can create pull up peaks in
other colors). See Signal
intensity is too high or
saturated on page 166.
Decrease sample concentration during PCR or

when preparing samples for electrophoresis.
Degraded PCR products.
Repeat PCR.
Stutter peaks.
See Identifying stutter products in microsatellite

analysis on page 113.
Incomplete restriction or
ligation (AFLP applications only).
Extract the DNA again and repeat the

restriction-ligation.
Sample is not denatured.
Make sure the samples are heated at 95C for

3 to 5 minutes, then immediately placed on ice for
2 to 3 minutes before loading.
Hairpin secondary structures

are present in PCR primers.
Non-specific primer peaks.
172
Primer/dimer peaks.
See Primer design guidelines on page 29.
Artifact peaks.
See Pull-up peaks from a sample appear in the

red or orange dye signal, and are detected as
size-standard peaks due to over-saturation of
sample-peak signal. on page 185.
Sample or reagent
contamination.
Use fresh sample or reagent.
Contamination with exogenous

DNA.
Use appropriate techniques to avoid introducing

foreign DNA during laboratory handling.
Renaturation of denatured
samples.
Load the sample immediately following

denaturation, or store it on ice until ready.
Symptom
Possible cause
11
Action
Pull-up peaks
Incorrect or poor-quality matrix

or spectral calibration.
Run a new spectral calibration.
Wrong matrix or spectral use for

analysis of 310 instrument data.
Reanalyze with the correct matrix in the

GeneMapper Software.
Offscale, saturated signal in

primary peak caused by high
sample concentration.
Polymer on instrument >7 days,

degraded polymer
contaminants.
Perform warm water wash(es) and replace

polymer.
Edit the instrument protocol to specify the correct

spectral calibration.
If the problem persists, replace the capillary array.
173
11
Symptom
Possible cause
Action
Data spikes
Bubbles, dried polymer, or dust

in the capillary array migrate
past the camera.
1. Flush the water trap. Refer to the instrument

user guide for information.
2. Check for bubbles and run the bubble wizard if
any are visible. Clean all connections and tubing
around the instrument pump.
3. Check the polymer bottle, the area around the
pump lines, and the array port for crystals. If
present, warm the polymer gently to 30C with
gentle mixing, then refill the pump and array
with the polymer.
4. If the problem persists, perform a water wash
and replace the polymer.
174
Symptom
Possible cause
11
Action
Split peaks
Plus A and/or minus A peaks

due to:
Repeat the experiment with a lower initial

template concentration.
Too many fragments for

A-addition.
Modify the experiment to:
Suboptimal PCR conditions

and/or suboptimal primer
design.
Increase addition of A:
Add a final extension step of 60 minutes at
72C.
Increase Mg2+ concentration
Use ABI PRISM Tailed Primer Pairs
Remove A by enzymatic treatment (T4 DNA
polymerase)
For more information, see Avoiding incomplete 3'
A nucleotide addition on page 34.
Many small extraneous peaks

appearing next to a
high-intensity peak
Background signal is above

Minimum Peak Height value.
Adjust the setting in analysis method.
High sample concentration.

(Extraneous peaks represent
non-specific DNA comigrating
with main fragment peak.)
Dilute the sample.
Sample concentration is too

high.
Decrease the injection time or injection voltage.

175
11
PCR troubleshooting
PCR troubleshooting
Symptom
Poor priming resulting in
weak signal
Possible Cause
Melting temperature is too
low due to low G+C content
and/or short primer length.
Action
Evaluate primer design.
Secondary structure of the

primer, particularly at the 3'
end.
Secondary structure of the
template in the region of
hybridization.
Amplified DNA
concentration is lower than
expected
PCR inhibition
Insufficient [F]dNTPs added

to PCR reaction.
Reamplify using more [F]dNTPs or examine the efficiency

of the PCR.
Amplification cycle setting is

too low.
Add 3 to 5 cycles.
Low MgCl2 concentration.
Increase the MgCl2 concentration.
Low affinity of the primer to

the template.
Decrease the annealing temperature 2 to 3C at a time.

Background signal may increase.
Low sample concentration.
Increase sample concentration.
Inhibitors in template.
Purify template (see Purifying DNA on page 56).
Thermal cycler malfunction.
Troubleshoot the thermal cycler problem. Refer to the

thermal cycler user guide for information.
PCR reagents are

contaminated or expired.
Use fresh PCR reagents.
Degraded primers.
Store unused primers at 15 to 25C. Do not expose

fluorescent dye-labeled primers to light for long periods of
time.
Sample contains
hemoglobin, heparin,
polyphenol (plant),
polysaccharides.
Dilute the sample before amplification to reduce the

amount of PCR inhibitors.
Extraction introduced
inhibitors (chloroform,
phenol, EDTA, detergents
(SDS), xylol, ethanol,
bromophenol blue).
Contamination with
exogenous DNA
176
Carryover.
Use appropriate techniques to avoid introducing foreign

DNA during laboratory handling. For more information,
see Avoiding contamination on page 64,
PCR troubleshooting
Symptom
Poor amplification,
non-specific amplification
Possible Cause
Poor-quality or degraded
DNA template.
11
Action
Use fresh template.
Run an agarose gel to check sample concentration and
quality.
If DNA is stored in water, check water purity.
Poor amplification, nonspecific amplification in

restriction-ligation
experiments (AFLP only)
Insufficient or excess
template DNA.
Use recommended amount of template DNA. Run an

agarose gel to check sample concentration and quality.
PCR inhibitors in the DNA

sample (binding proteins,
salts that carry over from
poor DNA extractions).
Use different extraction procedures.
Incorrect thermal cycling

parameters.
Check protocol for correct thermal cycling parameters.
Incorrect pH.
Use correct concentration of DNA and buffer.
Tubes loose in the thermal

cycler.
Push reaction tubes firmly into contact with block before

first cycle.
Third-party or non-PCR tube

type used.
Use GeneAmp Thin-Walled Reaction Tubes with Caps

with Thermo Fisher Scientific thermal cyclers.
Primer concentration too

low.
Use recommended primer concentration.
Primer design.
See Non-specific amplification on page 32.
Incomplete restrictionligation (in experiments

involving restriction-ligation)
due to insufficient or
insufficiently active ligase.
1. Test the ligase activity with control DNA.

2. Repeat restriction-ligation with a higher concentration
of ligase (in Weiss units).
Note: 1 Weiss unit = 67 cohesive-end ligation units
If the problem persists, repeat the restriction-ligation
with fresh enzymes and buffer. Use an agarose gel to
check the reaction results.
Refer to the AFLP Plant and Microbial Protocols for
more information (see AFLP applications on
page 200).
TE0.1 is buffer not properly

made, or contains too much
EDTA.
Add the appropriate amount of MgCl2 solution to amplified

reaction. Remake the TE0.1 buffer.
Insufficient enzyme activity.
Repeat the experiment with the recommended amount of

restriction enzyme, ligase, and AmpliTaq DNA
Polymerase.
Note: 1 Weiss unit = 67 cohesive-end ligation units.
Hairpin secondary
structures in PCR primers
Primer design.
177
11
Irregular baseline troubleshooting
Symptom
Possible Cause
Primer/dimer formation
MgCl2 concentration.
Action
See Optimizing PCR on page 55.
Annealing temperature in
the PCR.
Primer design.
Too much primer added to

reaction.
Prepare new reaction.
Primer over-amplification
due to insufficient or
poor-quality template.
Prepare new reaction.

Excessivenoiseoranelevatedbaselineaffectsbothsizingandgenotypingresults.
Symptom
Constant elevated signal
in raw data
Possible Cause
Action
Baseline waterfall
Waterfall (most
common on 310
instruments)
Contamination from marker

pen ink (if you used a marker
to label the plate or the heat
seal).
178
Prepare new plate.
Symptom
(continued)
Constant elevated signal
in raw data
Waterfall (most
common on 310
instruments)
Possible Cause
11
Action
Contamination from water

used to make buffer, wash
reservoirs/septa, for sample
injection, for any sampleprep steps, or for water
wash.
Use fresh water.
Instrument contamination.
Refer to the instrument user guide for troubleshooting

information.
Improperly filled/leaky
connections, tubing, or
polymer block.
Spectral/matrix calibration
issue.
Use correct matrix standard (see Dye sets and matrix

standards on page 41).
Specify the correct dye set in the instrument protocol.
Ensure the correct dye set was selected in spectral
calibration.
Apply the correct matrix file (310 instruments only).
Noisy baseline
Polymer on instrument
>7 days, polymer degraded
or precipitated.
Perform warm water wash(es) and replace polymer. If the

problem persists, replace the array.
Arcing/electronic noise.
Remove bubbles. Refer to the instrument user guide for

information.
Amplification of non-specific
products during PCR.
Degraded or incorrectly
stored Hi-Di Formamide
can cause low signal and
degraded products.
Use fresh, properly stored Hi-Di Formamide. See HiDi Formamide storage on page 82.
Capillary is contaminated.
Perform a water wash.
Weak or low signals and/or

an elevated baseline.
See Irregular signal intensity troubleshooting on

page 164.
Salt preferentially injects smaller fragments and inhibits

injection of larger fragments, so the majority of salt may
have been injected in the first injection.
If signal intensity does not increase, see Desalting on
page 190.
Adequate signal strength

with noisy data
Electrical noise
Contact Thermo Fisher Scientific.
Secondary hybridization site

is present on primer, which
results in many extra peaks.
Evaluate primer design.
Impure primer. You may see

a shadow sequence of N-1.
HPLC-purify the primer.
179
11
Instrumentation troubleshooting
Somedataqualityissuesarenotsamplerelated,butarecausedbysettingsor
conditionsusedfortheinstrumentrun.
Note: ThecolorofthecurrenttracevariesbetweenversionsofDataCollection
Software.
Symptom
Low or fluctuating current
Possible Cause
Action
Expired or incorrectly stored

buffer and/or polymer.
Use fresh buffer and polymer.
Bubbles in polymer.

information.
Anode buffer jar (3130

Series, 3730 Series, or 310
instruments) buffer is not
above required level.
Fill the anode buffer jar to the required level.
ABC (3500 Series

instruments) buffer is not
above required level.
For opened containers: Pipet buffer from the overflow

chamber to the main chamber.
Fluctuating current
Arcing caused by bubbles.

information.
Current too high
Decomposition of urea in the

polymer.
Use fresh polymer.
Incorrect buffer formulation

(most likely too
concentrated) (not applicable
to 3500 Series instruments).
Use correctly prepared buffer.
Arcing to conductive surface

on the instrument.
Ensure that the ambient temperature is 15 to 30C and the

humidity is <80%. Check for excessive condensation on the
instrument.
180
For unopened containers: Invert the

ABC, then tilt it slightly to make
sure most of the buffer is in the
larger side of the container. There
should be less than 1 mL of the
buffer remaining in the smaller side
of the container.
Symptom
Drop-off of current signal
Possible Cause
11
Action
Data Collection Software EPT view (current trace is blue)
Current signal should be

horizontal
Instrument has stopped

running and red light is on.
There are black marks
inside the instrument.
Air bubble in lower polymer

block.

information.
Clogged capillary (caused by

sample overloading).
Flush the capillary array with polymer.
Arcing to conductive surface

on the instrument.
Ensure that the ambient temperature is 15 to 30C and the

humidity is <80%. Check for excessive condensation on the
instrument.
Arcing caused by bubbles in

polymer.
Remove bubbles from polymer.
GeneMapper Software EPT view (current trace is green)
Current signal shape should resemble

the example shown in Examine the
sample info, raw data, and EPT trace on
page 154
181
11
Sizing or Size Quality (SQ) troubleshooting

Viewing the
size-standard
definition
Sizingissuescanoccurifthepeaksdetecteddonotmatchthepeakslistedinthe
sizestandarddefinition,forexample,ifadditionalpeaksaredetectedassizestandard
peaks,orifsizestandardpeaksarenotdetected.
Toviewthepeaksdetectedinthesizestandardandthepeakslistedinthe
sizestandarddefinitionforasample,selectthesample,thenclick (AnalysisSize
MatchEditor)toviewthesizinginformationfortheselectedsample(s).
Iftheexpectedpeaksarenotdetected,yourfirstcourseofactionshouldbeto
determinethecauseofthepeakdetectionissueandresolvetheissue.
Ifyouwanttousethedataevenifthesizestandarddataisoflowerquality,youcan
modifythesizestandarddefinition(describedbelow)toeliminateoraddpeaksto
improvethesizestandardqualityresult.
Note: Datafor
SQsamplesisviewableonlyintheRawDataview.
FormoreinformationontechniquesforimprovingsizingaccuracyonThermoFisher
Scientificgeneticanalyzers,refertoRosenblumetal.(1997)andWenzetal.(1998).
Modifying the
size-standard
definition
1. IntheProjectWindow,selectToolsGeneMapperManager.
2. SelecttheSizeStandardtab,thenselectthesizestandarddefinitionusedto
analyzethedata.
3. SelectSaveAsandnamethenewsizestandarddefinition.
182
11
4. Openthenewsizestandarddefinitionandaddorremovepeaksasneeded.
Troubleshooting
Symptom
SQ
or
and...
Possible Cause
No sample plot is displayed for a

sample with the error No sizing
data
A plot is not displayed if sizing

fails.
Size Match Editor does not

display peak data
Incorrect Size Standard Dye

specified in size-standard
definition.
The fragment sizes of the sizestandard definition do not match

the positions of the detected
peaks.
Action
See Sizing or Size Quality (SQ) troubleshooting
on page 182.
Note: You can view data for failed sizing in the Raw
view (see Examine the sample info, raw data, and
EPT trace on page 154).
The Peak Amplitude Threshold

of the dye color associated with
the size standard is set too high
or low in the analysis method.
Verify that the correct dye and fragment sizes are

specified in the Size Standard definition.
Select, modify, or create correct size-standard
definition as needed (see Modifying the
size-standard definition on page 182).
Adjust the analysis method so that the peak

detection threshold associated is greater than the
height of the noise signal. See GeneMapper
Software peak detection settings on page 94.
IMPORTANT! We do not recommend decreasing
the threshold below 50 RFU.
Expired or degraded size

standard.
Use fresh size standard.
183
11
Symptom
SQ
or
and...

(continued)
Possible Cause
Action
Incorrect concentration of size

standard in sample loading
reagent.
Increase the concentration of size standard added

to subsequent runs.
Size-standard peaks are not

migrating as expected during a
normal run time.
See Migration troubleshooting on page 168.
Incorrect injection settings (for

example, the injection time is
too short).
Review the injection settings of the run module for

errors.

injection.
Smaller size-standard
fragments are not labeled
Size standard peak and primer

peak are in the same read
region (see figure below).
Modify the size standard-definition and remove the

size-standard peak that overlaps with the primer
peak.
Change the analysis range in the Advanced Peak
Detection Algorithm field in the Peak Detector tab
of the analysis method. Select Partial Range and
select a starting data point that eliminates the
primer peaks from analysis.
Size-standard peaks labeled in

primer read region
Larger size-standard peaks are

not present in trace
184
Size-standard definition
modified to eliminate peaks
labeled in primer read region
Run time too short.
Increase run time.
Late start caused by a reagent

issue, a blocked capillary, or
high sample concentration.
Use fresh polymer.
A non-Thermo Fisher Scientific

size standard was used.
Use a Thermo Fisher Scientific size standard.
Symptom
SQ
or
and...
Possible Cause
11
Action
Extra peaks in size-standard

trace
Pull-up peaks from a sample

appear in the red or orange dye
signal, and are detected as
size-standard peaks due to
over-saturation of sample-peak
signal.
Spectral calibration performed

with the incorrect matrix
standard for the dye set.
Perform a spectral calibration with the correct

matrix standard for the dye set (see Table18 on
page 86).
Spectral calibration is from a

different array or has not been
run within the last 3 months.
If the peak heights that cause the pull-up peak are

not near the saturation limits of the instrument,
repeat the spectral calibration.
The signal in a neighboring

capillary is very strong and
creating a bleed-through peak.
Decrease the sample concentration.
Degraded size standard. A size

standard can be degraded by
sitting at room temperature for
>24 hours or using improperly
stored Hi-Di Formamide.
Use fresh size standard and fresh, properly stored

Hi-Di Formamide. See Hi-Di Formamide
storage on page 82.
Wrong size-standard definition

was used for the analysis.
Re-analyze with correct size-standard definition.
Degraded or incorrectly stored

Hi-Di Formamide.

Decrease the injection time.
185
11
Symptom
SQ
or
and...
Sizing failures occur in a regular

pattern (the same wells fail
repeatedly)
Possible Cause
Action
Electrophoresis or pipetting
error.
Refer to the instrument user guide for information

on troubleshooting capillaries/arrays.
Defective capillaries/arrays.
Autosampler is misaligned.
Noise peaks are detected as

size-standard peaks
Noise
peaks
Contaminated sample.
Prepare new sample.

The Peak Amplitude Threshold

of the dye color associated with
the size standard is set too high
or low in the analysis method.
Adjust the analysis method so that the peak

detection threshold associated is greater than the
height of the noise signal. See GeneMapper
Software peak detection settings on page 94.
Note: You can adjust the threshold if you want to
examine the data. However, we recommend that
you rerun with increased size-standard
concentration.
Size call inaccurate for known

DNA sample
186
Incorrect size standard was

added to sample.
Repeat with the correct size standard.
Size standard peaks are not

sized correctly.
Examine the size standard peaks and compare the

sizes to the size standard definition. See Viewing
the size-standard definition on page 182.
Migration issues.
GeneMapper Software troubleshooting
11

Someproblemswithdatacanbecausedbythesettingsusedtoanalyzethedata.If
peakheight,morphology,andnumberofexpectedpeaksareacceptable,butthe
samplefailssizing,itmaybecausedbyanalysismethodsettingsthatarenot
optimizedforyourapplication.
Foradditionaltroubleshootinginformation,refertotheGeneMapperSoftwareHelp
andtheGeneMapperSoftwareReferenceandTroubleshootingGuide(Pub.no.4403673).
Symptom
Possible Cause
Action
Generic message - the error

for each sample may be
different.
View the Info tab (see Examine the sample info, raw data,
and EPT trace on page 154).
The bin set in the analysis

method does not match the
panel used for analysis.
Modify the Bin Set selection on the Allele tab in the

analysis method.
GeneMapper Software
error message
Error Message: The bin set

in the analysis method does
not match the panel used for
analysis.
For information on resolving the error, refer to the

GeneMapper Software Reference and Troubleshooting
Guide.
187
11
Symptom
Possible Cause
Action
al? label
or alleles are not falling
within bins
Allele not labeled
Allele is migrating at a
different rate than expected.
Bin is not defined for the

allele.
Modify the binset to define the allele.
Peak is detected as stutter.
Decrease the Stutter ratio in the analysis method.

If the problem persists, mask the stutter peak by labeling
the allele as a mono peak: In the Panel Manager, change
the marker repeat # to 1 for the marker in question.
Data not sorted by name
Peak intensity is below the

Peak Amplitude Threshold in
the analysis method.
Adjust the Peak Amplitude Threshold in the analysis

method.
The default setting in

GeneMapper Software
sorts the data by status
rather than sample name.
To resort the data:

Select EditSort, or
Shift+click the column header to sort by
Clicking once sorts in ascending order, clicking twice sorts
in descending order.
When adding samples to a

project, the expected data
files are not listed in the Add
Samples to Project dialog
box
Data was not collected as

expected.
Ensure the following on the Data Collection Software

computer:
The instrument completed the run(s) and data are
visible in Data Collection Software.
Sample files were extracted successfully (3130 Series
or 3730 Series instruments).
The run folder was created and saved on the
instrument computer.
The correct number of *.fsa sample files were created
within the run folder.
188
Symptom
Possible Cause
11
Action
Genotypes tab is grayed
No panel specified before

analysis.
Specify a panel in the Project window before analysis.
The software is detecting

two separate peaks as one.
Adjust analysis method Peak Detector settings:
Two peaks do not separate

and are detected as one
peak
Polynomial degree:
A higher setting increases the sensitivity of the curvefitting process. A lower setting decreases it.
Peak Window size value:
A lower setting increases the sensitivity of the curvefitting process. A higher setting decreases it.
Decrease the peak window size to 13 and re-analyze.
For more information, see GeneMapper Software peak
detection settings on page 94.
189
11
Preamplification gel troubleshooting
Preamplification gel troubleshooting

Symptom
Possible Cause
No DNA smear on agarose

gel (AFLP only)
Incomplete digestion of
DNA.
Action
Check that enzymes are not expired, incubation
temperature settings are correct, and so on. Redigest
DNA.
Desalting
Impact of high salt
concentration
Sampleshighinsaltresultinpoorinjectionsandlowsignalintensity.Youmaybeable
tocompensatefordecreasedsignalintensityby:
Reinjectingthesample.Saltpreferentiallyinjectssmallerfragmentsandinhibits
injectionoflargerfragments,sothemajorityofsaltmayhavebeeninjectedinthe
firstinjection.
Increasingtheinjectiontimeand/orinjectionvoltage.
Ifreinjectingandincreasingtheinjectionparametersdoesnotimprovesignalintensity,
desaltand/orconcentratethesamples.
Donotincreasesampleconcentrationbyevaporatingthesampleswithoutperforming
adesaltingstep.Doingsoincreasesthesaltconcentrationandpreventscomplete
denaturationoftheDNAwhichcausesdecreasedsignalstrength.
Eliminating salt
concentration as
the cause
Todetermineifsaltconcentrationiscausinglowsignalintensity,runthesizestandard
alone(seeRunningcontrolstoisolateaproblemonpage 156).Ifyousee:
Resolutionlosswiththesizestandard,troubleshootinstrument/reagentissue.
Refertotheinstrumentuserguideforinformation.
Noresolutionlosswiththesizestandard,addsampletothewellcontainingthe
sizestandardandrunagain.Ifresolutiondecreases,desaltthesample.
Desalting
Todesaltyoursample(s),tryoneofthefollowing:
NucAwaySpinColumnsNucAwaySpinColumnsremoveunincorporated
nucleotidesandsaltsafterprobesynthesisreactions.Rehydratethecolumn,
centrifugetoremovetheinterstitialfluid,addthesampletothetopofthecolumn,
andcentrifugeagain.
AmiconCentricon100Microconcentrator(orCentricon30forfragmentssmaller
than130bp)
EthanolprecipitationofthepooledPCRproduct,followedbyresuspensionin
distilled,deionizedwater.RefertoMolecularcloning(Sambrook,Fritsch,and
Maniatis1989)forprotocols.
Sampledialysisonafiltermembrane:
a. FloataMilliporeVSfilter(MilliporePartno.VSWP02500),shinysideup,
ontopof50mLofdeionized,autoclavedwaterina50mLconicalplastic
tube.
b. Carefullyspot~15Lofsampleontopofthefilter,usinganappropriate
pipette.
190
Evaluating 310 Genetic Analyzer multicomponent matrix quality
11
c. Dialyzethesamplefor20minutes.
d. Usingapipette,verycarefullyremovethesampleanddilute.

Purpose of the
multicomponent
matrix
Factors affecting
matrix quality
Themulticomponentmatrixcompensatesfortheoverlapofdifferentdyecolorsby
subtractingout,ineachdyesdetectionrange,theportionofthesignaldueto
fluorescencefromotherdyes.
Reagentquality/freshness
Buffertypeandconcentration
Polymertype
Denaturingornondenaturingconditions
Runtemperature
When to create a
new matrix
Whenyoucreateamatrix,youmustruneachdyematrixstandardseparatelyto
determinetheproportionalamountoffluorescencethatisemittedinallfourdetection
regions.
Becausetheemissionspectraofthedyesvarywiththephysicalenvironment(suchas
thepHorpolymertypeandconcentration),createanewmatrixifrunconditions
change.
Ifyouobserveanyofthesymptomslistedinthetableonthenextpage,createanew
matrix.Youcanapplythenewmatrixtooldsamplesandreanalyzethedata.
Virtual Filter Set C
MatrixfilesmadeforVirtualFilterSetCareespeciallysusceptibletominorchangesin
runconditionsbecauseoftheemissionmaximumof6FAMdye(therecommended
bluedisplayingdyeforthisfilterset):
Itisveryclosetothelaserwavelengthof514.5nm.Thus,thewindowforcollected
bluelightintensitydataisoffsettolongerwavelengthsanddoesnotcontainthe
emissionmaximumof6FAMdye.
ItisalsoveryclosetothedetectionregionforthegreendisplayingTETdye.
IfyouareusingVirtualFilterSetC,watchforevidenceofmatrixproblemsandcreate
anewmatrixassoonasproblemsappear
Identifying matrix
problems
Apoororincorrectmatrixresultsintoomuchortoolittlesubtractionofdyespectral
overlapduringdataanalysis.Eachcausesarecognizableelectropherogramanomaly:
Bleedthroughpeaks,alsocalledpullups,arecausedbytoolittlesubtraction
Bleedthroughpeaksaresmallpeaksofonecolorlyingdirectlyunderalargepeak
ofanothercoloreventhoughthereisnoPCRproductcorrespondingtothe
smallerpeak.
Elevatedinterpeakbaselineiscausedbytoomuchsubtraction
Thetableonthenextpagecontainsexamplesofthesymptomslistedabove.
191
11
Symptom
Possible Cause
Action
Pull-up peaks (too little

matrix subtraction)
The matrix was made with the wrong

dyes or filter set.
Create a new matrix with the correct dye and

filter set. See Dye sets on page 41.
The signal from a large peak is off-scale

because of sample overloading. In the
raw data, the peak showing pull-up is
off-scale.
Keep peak heights between approximately 150

and 4000 RFU. If sample data is off-scale, do
one of the following:
Rerun the samples using a shorter injection
time.
Dilute and rerun the samples.
Create a new matrix with the correct dye
and filter set. See Dye sets on page 41.
Elevated interpeak baseline

(too much matrix
subtraction)
Too much matrix subtraction
192
Create a new matrix.
Thermalcyclersandaccessories........................................ 193
Geneticanalyzersandconsumables .................................... 194
GeneScansizestandards ............................................ 197
Matrixstandardsforspectralcalibration ................................ 197
Installationstandards................................................. 197
Reagentkits ......................................................... 198
Otherusersuppliedmaterials ......................................... 198
Thermal cyclers and accessories

Item
Veriti 96-Well Thermal Cycler
GeneAmp PCR System 9700 with the Silver 96-Well Block
GeneAmp PCR System 9700 with the Gold-plated Silver 96-Well Block
Silver 96-Well Sample Block
Gold-plated Silver 96-Well Sample Block
Source
4375786
N8050001
4314878
N8050251
4314443
MicroAmp
Autoclaved Reaction Tubes with Caps, 0.2 mL
N8010612
GeneAmp
Autoclaved Thin-walled Reaction Tubes with Domed Caps, 2000 tubes
N8010537
GeneAmp
Autoclaved Thin-walled Reaction Tubes with Domed Caps, 1000 tubes
N8010611
MicroAmp 96-Well Tray
N8010541
MicroAmp Reaction Tube with Cap, 0.2-mL
N8010540
MicroAmp 8-Tube Strip, 0.2-mL
N8010580
MicroAmp
8-Cap Strip
N8010535
MicroAmp
96-Well Tray/Retainer Set
MicroAmp
96-Well Base
MicroAmp
Clear Adhesive Film
4306311
MicroAmp
Optical Adhesive Film
4311971
MicroAmp Optical 96-Well Reaction Plate
403081
N8010531
N8010560
193
Genetic analyzers and consumables

Item
Source
Applied Biosystems 3500 or 3500xL Genetic Analyzer

Applied
Biosystems
Contact your local

Thermo Fisher Scientific
sales representative
3500xL Genetic Analyzer
3130 or 3130xl Genetic Analyzer

3730 or 3730xl DNA Analyzer
310 Genetic Analyzer
3500/3500xL Analyzer materials
Anode Buffer Container (ABC)
4393927
Cathode Buffer Container (CBC)
4408256
Septa Cathode Buffer Container, 3500 Series
4410715
Conditioning Reagent
4393718
Capillary Array, 36 cm, 8 Capillary, 3500 Genetic Analyzers
4404683
4404685
Capillary Array, 36 cm, 24 Capillary, 3500xL Genetic Analyzers
4404687
Capillary Array, 50 cm, 24 Capillary, 3500xL Genetic Analyzers
4404689
GeneScan Size Standards
See GeneScan size

standards on page 197.
Matrix Standard Kits
See Matrix standards

for spectral calibration
on page 197.
POP-4 Polymer (960 Samples), 3500/3500xL Genetic Analyzers
4393710
4393715
4393712
4393717
POP-7
Polymer (960 Samples), 3500/3500xL Genetic Analyzers
4393710
POP-7
Polymer (384 Samples), 3500/3500xL Genetic Analyzers
4393715
8-Tube Retainer and Base Set (Standard) for 3500/3500xL Genetic Analyzers
4410231
8-Strip Septa for 3500/3500xL Genetic Analyzers
4410701
96-well Retainer and Base Set (Standard) 3500/3500xL Genetic Analyzers
4410228
96-Well Septa for 3500/3500xL Genetic Analyzers
4412614
3730/3730xl Analyzer materials

Capillary Array, 50 cm, 48 Capillary, 3730 for Genetic Analyzers
4331250
Capillary Array, 36 cm, 48 Capillary, 3730 for Genetic Analyzers
4331247
Capillary Array, 50 cm, 96 Capillary, 3730xl for Genetic Analyzers
4331246
Capillary Array, 36 cm, 96 Capillary, 3730xl for Genetic Analyzers
4331244
GeneScan
194
Size Standards
See GeneScan size

Item
Source
on page 197.
POP-7 Polymer (28 mL), 3730/3730xl Genetic Analyzers
4363929
POP-7
Polymer (140 mL), 3730/3730xl Genetic Analyzers
4335615
POP-7
Polymer (280 mL), 3730/3730xl Genetic Analyzers
4363935
POP-7 Polymer (840 mL), 3730/3730xl Genetic Analyzers
4335611
MicroAmp Optical 96-Well Reaction Plate
N8010560
Hi-Di Formamide
4311320
Reservoir Septa
4315932
Running Buffer, 10
402824
96-Well Plate Septa
4315933
3130/3130xl Analyzer materials

96-Well Plate Septa
4315933
Reservoir Septa
4315932
4333465
4333466
4333464
4333463
Capillary Array, 80 cm, 16 Capillary, 3130xl Genetic Analyzers
4319899
4315930
4315931
Capillary Array, 22cm, 16 Capillary, 3130xl Genetic Analyzers
4319898
See GeneScan size


on page 197.
POP-4 Polymer (7000 L), 3130/3300xl Genetic Analyzers
4352755
4363752
4352757
POP-6
Polymer (3500 L), 3130/3300xl Genetic Analyzers
4363783
POP-7
4352759
POP-7
4363785
Running Buffer, 10
MicroAmp
Optical 96-Well Reaction Plate
402824
N8010560
3100/3100-Avant Genetic Analyzer Autosampler Plate Kit, 96-well
4316471
Hi-Di Formamide
4311320
310 Analyzer materials
195
Item
Source
Capillary Array, 47-cm, 310 DNA Analyzer capillary array
402839
Capillary Array, 61-cm, 310 DNA Analyzer capillary array
402840
96-well tray adaptor (for 9700 thermal cycler trays)
4305051
See GeneScan size


on page 197.
0.5 mL sample tray
5572
MicroAmp 8-tube strip, 0.2-mL
N8010580
MicroAmp 96-well base (holds 0.2-mL reaction tubes)
N8010531
MicroAmp
96-well full plate cover
N8010550
MicroAmp
96-well tray/retainer set
403081
POP-4
Polymer (5000 L), 310 Genetic Analyzers
402838
POP-6
Polymer (5000 L), 310 Genetic Analyzer
402837
Hi-Di
Formamide
4311320
Running Buffer, 10
4335643
Genetic Analyzer Septa Retainer Clips for 96-tube Sample Tray
402866
Genetic Analysis Sample Tubes (0.5-mL)
401957
Septa for 0.5-mL Sample Tubes
401956
For the Safety Data Sheet (SDS) of any chemical not distributed by Thermo Fisher Scientific, contact the chemical manufacturer. Before handling
any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions.
196
GeneScan size standards
GeneScan size standards

LIZ dye-labeled size
standards
(5-dye chemistry)
ROX dye-labeled size

standards
(4-dye chemistry)
Part no.
Part no.
GeneScan 120 LIZ
4324287
GeneScan 350 ROX
401735
GeneScan 500 LIZ
4322682
GeneScan 400HD ROX
402985
GeneScan 600 LIZ
4366589
GeneScan 500 ROX
401734
4408399
GeneScan
401098
GeneScan
GeneScan
600
LIZ
1200
v2.0
LIZ
1000
4379950
ROX
For denaturing and non-denaturing applications.

For non-denaturing applications only.
Matrix standards for spectral calibration

Dyesetshavebeentestedandoptimizedfortheinstrument,exceptwherenoted.
Dye Set part numbers
Instruments
DS-02
DS-20
DS-30
DS-31
DS-32
DS-33
DS-34
3500 Series
4323014
Not
tested
4345827
4345829
4345831
4345833
Not
tested
3730 Series
4323014
Not
tested
4345827
4345829
4345831
4345833
Not
tested
3130 Series
4323014
Not
tested
434445827
4345829
4345831
4345833
Not
tested
310
4323050
401114
401546,
402996
401546,
402996,
4313939
4312131
4318159
401546
Dye primer matrix standards.

We have tested this dye set but have not optimized for this instrument.
Installation standards
Installation standard
Source
For use with
DS-33 GeneScan Installation Standards
4376911
GeneScan 600 LIZ Size Standard v2.0

(6-FAM, VIC, NED, and PET dyes)
4330397
GeneScan 500 LIZ Size Standard

(6-FAM, VIC, NED, and PET dyes)
4316144
GeneScan 500 ROX

(6-FAM, HEX, and NED dyes)
197
Reagent kits
Reagent kits
Item
Source
AFLP Kits
AFLP I Selective Primer Kit
4303050
AFLP
II Selective Primer Kit
4303051
AFLP
EcoRI Ligation/Amplification Module
402941
AFLP
MseI Ligation/Amplification Module
402942
AFLP
Amplification Core Mix Module
402005
SNaPshot Kits
SNaPshot Multiplex Kit
Contact your local

representative
SNaPshot Primer Focus Kit
4329538
GeneAmp EZ rTth RNA PCR Kit
N808-0178
GeneAmp
PCR Carryover Prevention Kit
N808-0068
AmpErase
UNG
N808-0096
Other user-supplied materials

Item
Hi-Di Formamide, 25-mL
Source
4311320
Aerosol resistant pipette tips
MLS
Microcentrifuge tubes
MLS
Pipettors
MLS
Tape, labeling
MLS
Tube, 50-mL Falcon
MLS
Tube decapper, autoclavable
MLS
Deionized water, PCR grade
MLS
Vortex
MLS
Millipore
VS filter
CEPH Individual 1347-02 Control DNA
Millipore Part no. VSWP

02500
403062
198
Thepublicationnumbersinthissectionareforthelatestproductversionsavailableat
thetimeofpublication.Fordocumentationfornewerproductversions,goto
www.lifetechnologies.com.
Instrument documentation
Publication
number
Document
Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (3500 Series Software v1.0)
Applied
Biosystems
3500/3500xL Specification Sheet
Applied
Biosystems
3730 DNA Analyzer Getting Started Guide
Applied
Biosystems
3730/3730xl DNA Analyzer Specification Sheet
Applied
Biosystems
3130/3130xl Genetic Analyzers Getting Started Guide
4401661
106SP11-01
Applied Biosystems 3130/3130xl Specification Sheet
4359476
106SP08-01
4352715
106SP07-03
310 Genetic Analyzer User Guide
4317588
310 Genetic Analyzer Specification Sheet
903565
Veriti
4375799
Thermal Cycler User Guide
GeneAmp
PCR System 9700 User Guide
4303481
GeneMapper Software documentation

Publication
number
Document
GeneMapper Software Getting Started Guide: AFLP System Analysis
4403620
GeneMapper Software Getting Started Guide: Loss of Heterozygosity (LOH) Analysis
4403621
GeneMapper
4403672
Software Getting Started Guide: Microsatellite Analysis
GeneMapper
SNaPshot
Software Getting Started Guide:
GeneMapper
Software Reference and Troubleshooting Guide
4403614
GeneMapper
Software Installation and Administration Guide v4.1
4403614
GeneMapper
Software Reference and Troubleshooting Guide v4.1
4403673
GeneMapper Software Quick Reference Guide v4.1
Kit Analysis
4403618
4403615
199

Peak Scanner Software documentation
Peak Scanner Software documentation

Publication
number
Document
Peak Scanner Software Reference Guide
4382253
Peak Scanner Software Quick Reference Card
4383719
Application documentation
Publication
number
Document
AFLP applications
GeneMapper Software Getting Started Guide: AFLP System Analysis
4403620
AFLP Microbial Fingerprinting Protocol
402977
AFLP Plant Mapping Protocol
4303146
Aneuploidy applications
Aneuploidy Detection by QF-PCR of STR Markers on the Applied Biosystems 3500xL Genetic Analyzer
106AP28-01
BAC applications
BAC Fingerprinting on the Applied Biosystems 3730/3730xl DNA Analyzer
107AP04-01
Sizing of Large DNA Fragments Generated by BAC Fingerprinting on Capillary Electrophoresis System
106AP25-01
HiCEP applications
High coverage gene expression profiling on the Applied Biosystems 3500xL Genetic Analyzer
CO15884
ISSR applications
ISSR Genotyping of Endangered Plants Using an Optimized Workflow
106AP31-01
LOH applications
GeneMapper Software Getting Started Guide: Loss of Heterozygosity (LOH) Analysis
Relative Fluorescent Quantitation on CapillaryElectrophoresis Systems: Screening for Loss of Heterozygosity
in Tumor Samples on the Applied Biosystems 3130 Series Genetic Analyzers with GeneMapper
Software v3.7
4403621
106AP15-01
Methylation applications
Cells-to-CpG Bisulfite Conversion Kit (50) Protocol
4448998
Advances in Capillary Electrophoresis-Based Methods for DNA Methylation Analysis
106BR14-01
Three Optimized Workflows for CpG Island Methylation Profiling
106AP24-01
Microsatellite applications
GeneMapper Software Getting Started Guide: Microsatellite Analysis
Fact sheet: Microsatellite Analysis on the Applied
Biosystems
4403672
3130 Series Genetic Analyzers
Uniparental disomy (UPD) analysis of chromosome 15
106MI61-01
CO18249
Relative fluorescence quantitation applications

Aneuploidy Detection by QF-PCR of STR Markers on the Applied Biosystems 3500xL Genetic Analyzer
200
106AP28-01

Obtain SDSs
Publication
number
Document
Relative Fluorescent Quantitation on Capillary Electrophoresis Systems: Screening for Loss of Heterozygosity
in Tumor Samples on the Applied Biosystems 3130 Series Genetic Analyzers with GeneMapper Software
v3.7
106AP15-01
SNP applications
GeneMapper Software Getting Started Guide: SNaPshot Kit Analysis
the SNaPshot
User Bulletin: Using

Multiplex System with the POP-7 Polymer on Applied
3730/3730xl DNA Analyzers and 3130/3130
4403618
Biosystems
4367258
SNaPshot Multiplex Kit Quick Reference Card
4323975
Demonstration of SNP Genotyping Using a Single Nucleotide Extension Method and the 3500 Series Genetic
Analyzer
CO12863
Using the SNaPshot Multiplex System with the POP-7 Polymer on Applied Biosystems 3730/3730xl
DNA Analyzers and 3130/3130xl Genetic Analyzers
4367258
Obtain SDSs
SafetyDataSheets(SDSs)areavailablefromwww.lifetechnologies.com/support.
Obtain support
Forthelatestservicesandsupportinformationforalllocations,goto:
www.lifetechnologies.com/support
Atthewebsite,youcan:
AccessworldwidetelephoneandfaxnumberstocontactTechnicalSupportand
Salesfacilities
Searchthroughfrequentlyaskedquestions(FAQs)
SubmitaquestiondirectlytoTechnicalSupport
Searchforuserdocuments,SDSs,vectormapsandsequences,applicationnotes,
formulations,handbooks,certificatesofanalysis,citations,andotherproduct
supportdocuments
Obtaininformationaboutcustomertraining
Downloadsoftwareupdatesandpatches
201

Obtain support
202
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205
References
206
Glossary
allele
Onespecificsequenceofalocus.Differentallelesofasinglelocuswillhaveslightly
differentDNAsequences.
amplicon
TheproductofaPCRreaction.Typically,anampliconisashortpieceofDNA.
association studies
Studiesthatinterrogateadensesetofmarkerstoidentifyassociationsbetweenthose
markersandthelociofinterest.Associationstudiesaremorepowerfulthanlinkage
mappingstudiesforthedetectionofweaksusceptibilityloci.
backcross
AgeneticcrosswhereanindividualfromtheF1generationismatedtoanindividual
withthegenotypeofoneoftheparents.
bin
InGeneMapperSoftware,theexpectedlocationofanindividualpeakorallele,
definedbyabasepairrangeandacolor.Youtypicallydefineabinforeachpossible
alleleassociatedwithamarker.
binset
InGeneMapperSoftware,asetofbinsthatyouspecifyintheanalysismethod.
contig
InBACfingerprinting,acontiguoussequenceofDNAcreatedbyassembling
overlappingsequencedfragmentsofachromosome.Agroupofclonesrepresenting
overlappingregionsofthegenome.
diploid
Havingtwocopiesofeverychromosome.
dye set
Thetermdyesetcorrespondsto:
Thephysicaldyesetusedforlabelingfragments.
Thesoftwareselectionyoumakethatidentifiesthedyecolorsinthedyeset,the
orderofthedyepeaksinthedyeset,andspectralanalysisparametersforthe
dyes.
electrophoresis
Theactofapplyinganelectricalfieldtoaporoussubstrateinordertoseparate
moleculesbysizeorshape.Forexample,nucleicacidslikeDNAandRNAare
negativelychargedandarethusattractedtoapositivechargeandwillmovetowardit
throughamatrixlikeapolymeroragarosegel.Thestrengthoftheelectricalfieldand
chargeonthemoleculesandthesizeoftheporesinthematrixdeterminethespeedat
whichdifferentmoleculesinthestartingmixturemigrate.Ingeneral,smaller
moleculesmigratefasterthanlargermoleculesbecausetheyarelessobstructedasthey
travelthroughthematrix.
emission spectrum
Theintensityofemittedlight(fluorescence)asafunctionofthewavelengthofthe
emittedlight.
207
Glossary
excitation efficiency
Theprobabilitythatitwillabsorblightofacertainwavelength,asapercentageofthe
probabilityofabsorptionatthewavelengthofmaximumabsorption.
excitation spectrum
Theintensityofemittedlightasafunctionofthewavelengthoftheexcitinglight.
filter set
Physicalfiltersthatseparatedyesignalsinoldergelbasedcapillaryelectrophoresis
instruments.NewerThermoFisherScientificinstrumentsuseadiffractiongrating.
fingerprint
Acharacteristicpatternofpeaksorbandsafteranamplificationordigestofgenomic
DNAthatcanbeusedtoidentifyanindividual.
genetic mapping
Analysisoftheprogenyfromgeneticcrossestodeterminetherelativepositionof
chromosomallocations.
Linkage mapping
Ameioticmappingtechniquewhichsearchesforlinkagebetweenamarkerandthe
diseasegenewithinseveralgenerationsofaffectedfamilies.Thesestudiesexaminethe
inheritanceofmarkersinbothaffectedandunaffectedmembersofthefamilyandthen
determinewhetherparticularmarkersarephysicallyclosetothediseasegeneof
interest.Linkagemappingcanbeusedtoscantheentiregenomerelativelyeasily.
ISSR PCR
IntersimplesequencerepeatPCR.SeeIntersimplesequencerepeat(ISSR)PCRon
page137.
locus
Alocationonachromosome.Thetermissometimesusedmorenarrowlytodescribe
thelocationofageneorgeneticmarker.(Plural:loci).SeealsoMarker.
marker
Anyobservablegeneticcharacteristic(forexample,gene,phenotype,microsatellite
sequence,SNP)thatcanbeusedtoidentifyageneticlocation.Tobeusefulingenetic
studies,amarkermustbepresentindifferentformstoallowresearcherstodistinguish
betweenindividuals.Onemarkerrepresentsonelocusandoneprimerpair.Defined
bysizerangeofexpectedallelesanddye(color)attachedtoprimer.
microsatellites
Highlyrepetitivesimplesequencerepeatsof2to7basepairs,alsocalledshorttandem
repeats(STRs).Microsatellitesfallintothebroadercategoryreferredtoasvariable
numberoftandemrepeats(VNTRs),whichalsoincludesminisatellites.
minisatellites
Highlyrepetitivesimplesequencerepeatsrangingfromapproximately10to30base
pairs.Minisatellitesfallintothebroadercategoryreferredtoasvariablenumberof
tandemrepeats(VNTRs),whichalsoincludesmicrosatellites.
panel
InGeneMapperSoftware,acollectionofexpectedsizerangesanddyecolors
(markers)associatedwitheachprimerpair.
phylogenetic studies
Studiestodetermineevolutionaryrelationshipsbetweenorganisms(forexample,
speciesorpopulations).
polymerase
Anenzymethatcatalyzespolymerization.DNAandRNApolymerasesbuild
singlestrandedDNAorRNA(respectively)fromfreenucleotides,usinganother
singlestrandedDNAorRNAasthetemplate.
polymorphic locus
Alocuswithmorethanoneallelethatiscommonlyfoundinapopulation.
208
Glossary
population study
Astudythattypesgeneticmarkersonalargepopulationtoidentifyassociations
betweenamarkerandaphenotype.
primer
AshortsinglestrandofDNAthatservesastheprimingsiteforDNApolymeraseina
PCRreaction.
pull-up peaks
Artifactpeaksinoneormoredyecolorsthatarecausedbyhighorsaturatedsignal
intensityinanotherdyecolor.
quantum yield
Theprobabilitythatadyeinitsexcitedstatewillemitaphotonasitdecaysbacktothe
groundstate.
restriction enzymes
EndonucleasesthatcleavethephosphatebackboneofdoublestrandedDNAathighly
specificsequencescalledrestrictionsites.
template
InPCR,thenucleicacidmoleculethatprovidesthesequencetoamplify.Forexample,
genomicDNAcanbethetemplateforaPCRreactionthatamplifiesaspecificregion
withinthegenome.
Variable Number
Tandem Repeat
(VNTR)
Anygeneswhoseallelescontaindifferentnumbersoftandemlyrepeated
oligonucleotidesequences.
209
Glossary
210
Index
Numerics
2720ThermalCycler 58
3Anucleotideaddition 33
310instrument
Seealsocapillaryelectrophoresis
dyesetsandmatrixstandards 86
matrix,overview 87
runmodule,updatingforGeneScanLIZSize
Standard 46
safetyinformation 68
signalintensityrange 77, 158
specifications 68
3130Seriesinstrument
runmodule,updatingforGeneScanLIZSize
Standard 48
runmodules,downloading 48
specifications 68
performance 70
runmodules 69
specifications 68
runmodule,downloading 48
runmodule,optimizingforGeneScan600LIZSize
Standard 48
runmodules 69
specifications 68
throughput 69
AccuPrimeTaqDNAPolymerase 22, 24
AccuPrimeTaqDNAPolymeraseHighFidelity 22
AFLP
advantages 126
analysismethod,GeneMapperSoftware 129
applications 127
dataanalysis 129
exampledata 129
experimentandprimerdesign
recommendations 127
genotypesinGeneMapperSoftware 130
instrumentandconsumable
recommendations 127
overview 125
PCRkits 198
principle 126
restrictionenzymes 128
workflow 128
agarosegel,usingfortroubleshooting 157
ambienttemperature,problemscausedby 160
amplification
SeealsoPCR
nonspecific 32
postamplificationmanipulations 33
selective 31
amplifiedfragmentlengthpolymorphism.SeeAFLP
AmpliTaqDNAPolymerase 22, 24
AmpliTaqDNAPolymerase,LD 22, 24
AmpliTaqGoldDNAPolymerase 23, 24
analysissoftware 89
aneuploidy,relativefluorescenceapplication 17
animalbreeding,microsatelliteapplication 16
animaltyping,microsatelliteapplication 16
annealingtemperature
optimizing 64
PCRparametersforunknown 61, 63
annealing,factorsaffecting 30
associationstudies 110
autosampler,problemscausedbymisalignment 161
211
Index
B
BACfingerprinting
applications 133
dataanalysis 73, 90, 159
recommendations 134
recommendations 134
overview 132
principle 133
workflow 135
bacterialartificialchromosome.SeeBACfingerprint
ing
baselinetroubleshooting 178
basepairingenergies 30, 31
bleedthroughpeaks.Seepulluppeaks
breeding 110
C
cancerprogressionanalysis 110
capillaryarray
foreachinstrument 68
problemscausedbydegradedorclogged 160
capillaryelectrophoresis
Seealso310instrument; 3130Series
instrument; 3500Seriesinstrument; 3730
Seriesinstrument
controlDNA 57
definition 18
factorsaffecting 82
injectiontime,optimizing 79
injectionvoltage,optimizing 80
optimizing 67
optimizingconditions 80
polymers 83
runmodules 46
runtime,optimizing 81
runvoltage,optimizing 81
signalintensity 77
spatialcalibration 84
spectralcalibration 84
troubleshooting 180
CE.Seecapillaryelectrophoresis
CEPH134702ControlDNA 57, 157
CNV,relativefluorescenceapplication 17
concentration
DNAtemplate 59
dNTP 59
212
enzyme 60
Mgion 59
sample 158
controls
CEPH134702ControlDNA 157
controlDNA,using 57
DNAtemplate 157
process 157
recommendationforfrequency 69, 156
runningtoisolateaproblem 156
CopyNumberVariation.SeeCNV
customprimerdesign 29
D
data
checkingquality 104, 153
electropherogramqualityrequirements 104
troubleshooting 153
dataanalysis
GeneMapperSoftware 89
PeakScannerSoftware 92
desalting 190
dinucleotiderepeats 115
DNAmethylation 149
DNApolymeraseenzymes
3Aaddition 33
characteristics 24
recommended 22
DNAtemplate
concentration 59
control 157
isolating 55
purifying 56
quantifying 56
storing 56
dyesets
dyecomponents 41
matrixstandardsfor 86
dyes
chemicalforms 36
emission 38
emissionmax 38
E
electropherogram,quality 104
electrophoresis.Seecapillaryelectrophoresis
EPTtrace
Index
examining 155
exampleofgoodquality 155
troubleshooting 180
errormessage,GeneMapperSoftware 154
evaluatingdata
electropherogram 104
stutter 113
excitationandemissionmax 38
experimentaldesignconsiderations 21
F
fingerprinting
AFLP 125
applications 17
BAC 132
HiCEP 136
ISSR 137
TRFLP 131
fluorescentlabeling 25
forensics.SeeHumanIdentification
fragmentanalysis
applications 16
definition 15
workflow 19
G
GeneAmpGoldFastPCRMasterMix 23
GeneAmpPCRSystem9700,384WellDual
Autolid 58
GeneAmpPCRSystem9700,Dual384well 58
GeneAmpPCRSystem9700,Dual96Well 58
GeneMapperSoftware
AFLPdefaultmethod 129
AFLPgenotypes 130
autoanalysis 91
errormessage 154
features 91
ISSRbins 139
microsatellitedefaultmethod 112
peakdetectionsettings 94
peakstartandendsettings 97
sizing 98
sizingmethods 100
SNaPshotdefaultmethod 123
troubleshooting 187
workflow 93
generalPCR,thermalcyclerparameters 61, 62
GeneScansizestandards.Seesizestandard
geneticanalyzer.See310instrument; 3130Series
instrument; 3500Seriesinstrument; 3730Se
riesinstrument
geneticdiversity
fingerprintingapplication 17
microsatelliteapplication 16
geneticmapsfornewspecies,fingerprinting
application 17
genomescans 110
guidelines
PCRcontamination 64
PCRparameters 63
primerdesign 29
sizecalling 90
H
hairpinsecondarystructures 31
HiCEP 136
applications 136
overview 136
principle 136
workflow 136
HID.SeeHumanIdentification
HiDiformamide
issuescausedbyimproperlystored 159
storageconditions 82
highcoverageexpressionprofiling.SeeHiCEP
hotstartPCR
description 60
enzyme 23
thermalcyclerparameters 60, 62
HumanIdentificationapplications.Gotolifetechnolo
gies.com
humidity,problemscausedby 161
I
injection
factorsaffecting 78
time,optimizing 79
voltage,optimizing 80
instruments
forusewithguide 13
platetypes 58
problemscausedby 160
RFUranges 158
runmodules 46
signalintensityranges 158
213
Index
troubleshooting 158, 159, 180

Intersimplesequencerepeat.SeeISSR
ionicstrengthofbuffer 78
ISSR
applications 138
creatingmultiplebins 139
dataanalysis 139
exampledata 140
recommendations 139
overview 137
principle 137
workflow 139
L
labeling,fluorescent 25
laboratorywater,problemscausedby
contamination 159
linkagegroupsamongcrosses,fingerprinting
application 17
linkagemapping
fragmentanalysis 16
microsatelliteanalysis 110
thermalcyclerparameters 62
LOH
relativefluorescenceapplications 17
LossofHeterozygosity.SeeLOH
lowcopyamplification,enzyme 22, 23
M
matrix
310instrument 191
standardsforspectralcalibration 86
methylationassessment 149
microbialgenemapping,fingerprinting
application 17
microsatelliteanalysis
advantages 108
applications 110
dataanalysis 112
recommendations 111
recommendations 111
overview 107
principle 108
214
sizestandard,GeneScan400HDROX 49
sizestandard,GeneScan600LIZ 111
stutter 113
troubleshooting 113
workflow 112
microsatelliteinstability,microsatellite
application 16, 146
migrationtroubleshooting 168
MLPA,relativefluorescenceapplications 17, 143
MLVA,microsatelliteapplication 16
multicomponentanalysis 26, 37
MultilocusVariantAnalysis.SeeMLVA
multiplexing
benefitsandlimitations 26
guidelines 28
poolingratios 27
software 28
strategies 27
troubleshooting 29
N
noise,troubleshooting 178
normalizationsizestandard 45
P
parentageanalysis 110
paternitytesting 110
pathogensubtyping,microsatelliteapplication 16
PCR
Seealsothermalcyclerparameters
3addition 33
carryover 65
contamination 64
controlDNA 57
DNAtemplate 55
general 61, 62
hotstart 60, 62
linkagemapping 62
maximizeyieldofspecificproducts 61, 63
nonspecificamplification 32
plates 58
primerdesign 29
primers,preparing 57
problemscausedbyexpiredreagents 159
products,storing 56
reactionvolume 58
Index
reagentconcentrations 59
selectiveamplification 31
setup 64
sidereactions 60
templatevolume 58
timerelease 61, 62
touchdown 61, 63
troubleshooting 176
XL 63
PCRworkareas 64
peakdetection
peakwindowsize 94
polynomialdegree 94
peakmorphologytroubleshooting 169
PeakScannerSoftware
features 92
overview 92
phylogeneticstudies 110
plantgenomemapping,fingerprintingapplication 17
planttyping,microsatelliteapplication 16
PlatinumMultiplexPCRMasterMix 23, 24
PlatinumPfxDNAPolymerase 23, 24
polymer
characteristics 83
handling 83
problemscausedbydegradedorexpired 159
types 83
polymeraseenzymes.SeeDNApolymeraseenzymes
polynomialdegree
peakdetection 94
varying 95
windowsizevalue 96
poolingratiosformultiplexing 27
POPpolymer.Seepolymer
populationgeneticsstudies 110
preamplificationtroubleshooting 190
primer/dimer
minimizing 62
primer/dimer,minimizing 60
primers
designguidelines 29
reconstituting 57
SNaPshot 123
tailing 34
Q
QFPCR,relativefluorescenceapplications 17, 143
QualitativeFluorescencePCR.SeeQFPCR
quantification
DNAtemplate 56
primers 57
QuantitativeMultiplexPCRofShortFluorescentFrag
ments.SeeQMPSF
R
rawdata
examine 154
example 155
reagenttroubleshooting 158, 159
RelativeFluorescenceQuantitation.SeeRFQ
Replicationerror 146
RER 146
resolution
definitionof 79
formula 79
troubleshooting 169
RFQ
applications 17, 144
dataanalysis 145
determiningrelativenumberofmolecules 146
determiningrelativequantities 145
recommendations 144
LOHworkflow 145
minimizingsignalintensityvariation 144
overview 143
peakheightversusarea 143
RFUrangesforinstruments 158
rTthDNApolymerase
description 24
thermalcyclerparameters 63
XL 24
runmodules 46
S
SafetyDataSheets(SDSs),obtaining 201
saltconcentration
desalting 190
impactonelectrophoresis 82
sample
concentration,problemscausedbyhigh 158
215
Index
contamination,problemscausedby 158
samplesaltconcentration.Seesaltconcentration
secondarystructure
primer 30
template 31
selectiveamplification 31
signalintensity
minimizingvariation 144
optimizing 77
rangeforeachinstrument 77, 158
relativeorderofdyes 38
troubleshooting 164
SingleNucleotidePolymorphism.SeeSNP
singleplexing 26
SizeMatchEditor,displaying 153
sizemethods,GeneMapperSoftware 100
sizestandards
function 42
GS1000ROX 51
GS120LIZ 44
GS1200LIZ 47
GS350ROX 49
GS400HDROX 49
GS500LIZ 44
GS500ROX 50
GS600LIZ 45
GS600LIZv2.0 45
modifyingdefinition 153
normalization 45
preparation 43
storage 43
troubleshooting 182
sizecallingguidelines 90
sizing
CubicSplinemethod 101
curve 100
GlobalSouthernmethod 103
howtheGeneMapperSoftwareperforms 98
LeastSquaresmethod 100
LocalSouthernmethod 102
methods 100
troubleshooting 153, 182
sizingquality
checking 153
troubleshooting 182
software,analysis 89
spatialcalibration,overview 84
spectralcalibration
216
matrixstandards 41
overview 84
splitpeaks,incomplete3Aaddition 33
SQ.Seesizingquality
standards
Seealsosizestandards 42
installstandardfortroubleshooting 157
matrixforspectralcalibration 86
stutter
identifying 113
troubleshooting 113
SuperScriptIIIReverseTranscriptase 23, 24
support,obtaining 201
T
tailingprimer 34
technicalsupport 201
template
DNA 56, 59
smallvolume 58
terminalrestrictionfragmentlengthpolymorphism.
SeeTRFLP
thermalcyclerparameters
generalPCR 61, 62
hotstart 60, 62
linkagemapping 62
optimizing 63
timereleasePCR 61, 62
touchdownPCR 61, 63
XLPCR 63
thermalcyclers
applications 58
specifications 58
timereleasePCR
enzyme 23
thermalcyclerparameters 61, 62
Tm
calculator 29
factorsaffecting 30
primer 29
touchdownPCR,thermalcyclerparameters 61, 63
training,informationon 201
TRFLP
applications 131
dataanalysis 132
recommendations 132
Index
recommendations 131
overview 131
principle 131
troubleshooting
agarosegel,running 157
ambienttemperature 160
autosampleralignment 161
baseline 178
bufferstrength 160
capillaryarray 160
capillaryelectrophoresis 180
CEPH134702ControlDNA 157
consumableissues 159
dataquality 153
EPTtrace 180
errormessage 187
extrapeaks 172
GeneMapperSoftware 187
instrumentissues 160, 180
isolatingtheproblem,controls 156
matrix 161
microsatelliteanalysis 113
migration 168
multiplexing 29
noise 178
PCR 176
peakmorphology 169
preamplificationgel 190
processcontrol 157
reagentissues 159
resolution 169
sampleissues 158
signalintensity 164
sizestandard 182
sizingandsizingquality 182
stutter 113
temperatureandhumidity 161
workflow 152
TthDNApolymerase 24
amplifiedDNA 65
PCRsetup 64
X
XLPCR,thermalcyclerparameters 63
V
Veriti384WellThermalCycler 58
Veriti96WellThermalCycler 58
VNTR 109
W
workarea
217
Index
218
Headquarters
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05 April 2014

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User Guide DNA Fragment Analysis by Capillary Electrophoresis (English) Manual Product Insert

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USER GUIDE

DNA Fragment Analysis

Publication Number 4474504

For Research Use Only. Not intended for use in diagnostic

2014 Thermo Fisher Scientific Inc. All rights reserved.

About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Introduction to Fragment Analysis . . . . . . . . . . . . . . . . . . 15

Fragment analysis versus sequencingwhat is the difference? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Experimental design considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Fluorescent labeling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Primer design guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DNA Fragment Analysis by Capillary Electrophoresis

Ordering custom primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Isolating, purifying, quantifying, and storing DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DNA Fragment Analysis by Capillary Electrophoresis

Using control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Reaction volumes and plate types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Preventing competing side reactions: hot-start PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Thermal cycling parameters 9700 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Optimizing thermal cycling parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Optimizing Capillary Electrophoresis . . . . . . . . . . . . . . . . . 67

DNA Fragment Analysis by Capillary Electrophoresis

3500 Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3730 Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3130 Series instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Optimizing electrokinetic injection parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Optimizing electrophoresis conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Other factors that affect electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Understanding spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

DNA Fragment Analysis by Capillary Electrophoresis

CHAPTER 5 Data Analysis with GeneMapper Software and Peak

GeneMapper Software features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

GeneMapper Software peak start and end settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Evaluating data quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Microsatellite Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Overview of microsatellite analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Single Nucleotide Polymorphism (SNP) Genotyping . . 119

Overview of SNP genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Amplified fragment length polymorphism (AFLP) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Terminal restriction fragment length polymorphism (T-RFLP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DNA Fragment Analysis by Capillary Electrophoresis

Instrument and consumable recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Bacterial Artificial Chromosome (BAC) fingerprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

High coverage expression profiling (HiCEP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Inter-simple sequence repeat (ISSR) PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Relative Fluorescence Quantitation (RFQ) . . . . . . . . . . 143

For more information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Additional Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 149

DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Troubleshooting workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Checking data quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Running controls to isolate a problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Reagent and consumable issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Instrument and ambient condition issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Symptoms you may observe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DNA Fragment Analysis by Capillary Electrophoresis

GeneMapper Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

GeneMapper Software troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Evaluating 310 Genetic Analyzer multicomponent matrix quality . . . . . . . . . . . . . . . . . . . . . . . . . .

Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

DNA Fragment Analysis by Capillary Electrophoresis