Analyzing Short Tandem

Repeats: PCR Products and

Interpretation
Lecture 7

Recap

PCR ON STR LOCI

DNA Profile

Short Tandem Repeat (STR) Markers

PCR primers anneal to unique sequences
bracketing the variable STR repeat region

PCR Product Size (bp)

The overall PCR product size is measured

Allelic Ladder

Fluorescent
dye

PCR product size generated

DNA template
containing STR marker

Forward
PCR primer

Reverse
PCR primer

GATA

GATA

GATA

Sample
#1

GATA

STR repeat region

TCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGAAG
ACAGGTGGATAGATAGATAGATAGATAGATAGATAGAT
AGATAGATAGATATCATTGAAAGACAAAACAGAGATGGA
TGATAGATACATGCTTACAGATGCACAC

Sample
#2

= 11 GATA repeats (11 is all that is reported)

of Forensic STR Markers on Human Chromosomes

13 Core U.S. STR
Loci

TPOX
D3S1358

D5S818

D8S1179

TH01

1997

VWA

FGA
D7S820
CSF1PO

8 STR loci overlap between U.S. and Europe

AMEL

D13S31
7

Sex-typing
D16S539 D18S51

D21S11

AMEL

Short Tandem Repeat (STR)

Typing
Fluorescent
dye-labeled
primer

STR Repeat Region

5

1 2

4 5

(Maternal)

7 8

(Paternal)

1 2

forward primer
hybridization region

4 5

GATA

(size in bp)
75.80.100.120.140.160.180.200.220.240.260..
1000
RFUs 500
6
139bp
8
147bp

DNA Separation and Detection

Section 1

SEPARATION OF THE DNA

PCR FRAGMENTS

(488 nm)

Excitation

LASER

A DNA Profile is Produced by Separating DNA Molecules by Size

and Dye Color

The labeled fragments are

separated (based on size) and
detected on a gel or capillary
electrophoresis instrument
~2 hours or less
Fragment size ranges from 100 - 350 base pairs

Peaks represent labeled DNA fragments separated by electrophoresis

This profile of peaks is unique for an individual a DNA type

Steps Involved in STR Genotyping

Data Collection
Matrix file
(spectral
calibration)
User-defined thresholds

Internal size
standard
Allelic ladder
sample

Color Separation

Peak Identification

GeneScan
software

Peak Sizing
Comparison to
Allelic Ladder

GeneMapperID
software

Genotyper
software

Peak Editing to
Remove Artifact
Calls

Genotype
Assignment to
Alleles
Data Review by
Analyst/Examiner

Confirmation of Results
by Second
Analyst/Examiner

Expert Systems
(e.g., FSS-i3,
TrueAllele)

Detection Thresholds
Thresholds are set to separate
signal from noise in other words,
are we confident that a peak is
real?
Signal peak height is measured in
relative fluorescence units (RFUs)
that are related to the amount of
DNA present in the sample loaded
onto the analysis instrument
Detection thresholds typically vary from 50 RFU to 200 RFU

STR Analysis

PEAK IDENTIFICATION

Thresholds for Measuring DNA Data

50 RFUs

Peak is
called
(deemed Detection (analytical)
reliable) threshold

Peak is NOT
called (deemed
unreliable)

Dependent on instrument
sensitivity
~50 RFU (relative fluorescence
units)
Impacted by instrument baseline
noise

Dropout (stochastic)
threshold
Dependent on biological sensitivity
~150-200 RFU
These thresholds for reliable
data are determined
through
Important
in mixture
validation
studies
interpretation
Baseline noise

Values shown for example purposes only

(should be based empirically on a labs
internal validation)

Peak Detection Thresholds

Peak reliable, can
be used for
inclusions

150 RFUs
Peak reliable, but
only used for
exclusions

50 RFUs
Peak not
considered
reliable

Interpretatio
n Threshold

Analytic
al
Thresho
ld
Baseline
Noise

DNA Data Quality

The raw DNA data itself does not have quality scores directly attached to it.
Only the STR allele designations are stored without an indication of
data quality.
Checks and balances exist in the entire system to try and ensure good
quality data.
Retesting of offender database sample is performed when a DNA database
hit is observed.

STR Analysis

PEAK SIZING

Peak Sizing with an Internal Size Standard

(a)
50
35

75 100

150
139160 200

250

350
300 340

Region depicted below

(b)

DNA
Size

400

500
450 490

250
200

165.05
bp
147.32
bp

160
150
139

100

DNA fragment
peaks in sample

DNA fragment peaks

are sized based on
the sizing curve
produced from the
points on the internal
size standard

Time (minutes)

Data
Point

Peak Color Separation and Sizing and STR

Typing
Allelic ladder

PCR-amplified sample
Data from CE
instrument (prior to
color separation and
peak sizing)
Genotyping allele bins

Color-separated and
sized allele peaks for
each locus

Color
separatio
n and
peak
sizing

(+/-0.5 bp around ladder allele)

Locus 1
Alleles (# repeats)

Color
separatio
n and
peak
sizing

10 11 12 13 14 15

Internal size standard

All ladder
alleles sized
using
internal size
standard

Genotype = 12, 14

Genotyping
performed by
comparing allelic
ladder to sample
results

Internal size standard

All sample
alleles sized
using
internal size
standard

Data Interpretation Issues

Artifact Peaks vs. Allele Peaks

Pull-Up
Stutter
N Peaks
Off Ladder Alleles
Tri-Alleles

Allelic Drop-Out
Degradation
Inhibition/Primer Binding Site Mutation

Mixture
Mutation

Error Peaks

PULL-UPS

Pull Ups: What appears may not be real

If Pull-Up is present and due to excess fluorescence, one may correct the
problem by Dilution of PCR Product.

Pull Ups: What appears may not be real

RFU Reporting
Threshold

The peaks that appear in the electropherogram may be excluded based on the
thresholds.
However exclusion based on thresholds may not be sufficient to remove the
error peaks.

Error Peaks

ALLELIC LADDER
INTERPRETATION

Comparison of Allelic Ladder to Samples to

Convert Size into Allele Repeat Number

+/- 0.5 bp bin

defined around each
allele

differenc
e = - 0.02
bp

difference
= + 0.05
bp

Overlay of all 4
colors
(including internal
size standard)

D3S1358TH01

D5S818

VWA
Amelogenin
(sex-typing)

D21S11

D7S820
D13S317

Penta E

D18S51

CSF1PO
D16S539

Penta D

blue panel

green
panel

yellow panel

D8S1179
TPOX

FGA

red panel
ILS600 DNA sizing standard
200
100
225 250 275
bp 120 140 160 180 bp

300
bp 325 350 375

400
500
425
450
475
bp
bp

D8S117
9

D21S11
(24 alleles)

(12 alleles)

TH01

D3S135
8

(10 alleles)

(8 alleles)

D19S43
3

D7S820

CSF1PO Blue panel

(10 alleles)

(10 alleles)

D13S31
7

D16S53
9

(8 alleles)

(9 alleles)

VWA

TPOX

(14 alleles)

(8 alleles)

Green panel

D2S133
8

(14 alleles)

Yellow
panel

D18S51
(23 alleles)

(15 alleles)

AMEL
(2 alleles)

D5S818
(10 alleles)

Red panel

FGA low

FGA
high

(19 alleles)

(9 alleles)

100
bp

150 bp
139bp
160
bp

LIZ-labeled GS500 DNA sizing

standard
200
250
bp
bp*

300
bp

340
bp

Orange panel
350
bp