Heat-Activatable Primers for Hot-Start UNIT 15.

9
PCR and Hot-Start One-Step RT-PCR:
Endpoint and Real-Time Experiments
Elena Hidalgo Ashrafi1 and Natasha Paul1
1
TriLink BioTechnologies, Inc., San Diego, California

ABSTRACT
Hot-start PCR is a technique that improves PCR performance by reducing nonspecific
amplification during the initial setup stages of the PCR. This unit describes hot-start
PCR protocols which utilize primers containing temperature-sensitive modifications.
The introduction of 4-oxo-tetradecyl (OXT) phosphotriester groups onto the 3 end of the
primer allows for primer-based hot-start PCR that is amenable for use in a number of PCR-
based applications. The protocols described in this unit utilize OXT-modified primers in
applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex
PCR, and one-step reverse-transcription PCR. This method is also advantageous for
instances where improved PCR specificity is desired and a hot-start polymerase suitable
for your application is not available. Curr. Protoc. Mol. Biol. 88:15.9.1-15.9.15. 
C 2009

by John Wiley & Sons, Inc.

Keywords: PCR r Hot Start PCR r primer dimer r mispriming r thermal cycler r
DNA polymerase r dNTPs r multiplex r fast-cycling PCR r RT-PCR r real-time PCR

INTRODUCTION
This unit provides reaction conditions for the use of chemically modified primers
(CleanAmp Turbo and Precision Primers) as a hot-start activation approach in PCR.
In this approach, improved specificity and efficiency of target amplicon formation are
achieved by the introduction of temperature-sensitive 4-oxo-tetradecyl (OXT) modifica-
tions onto the 3 terminal phosphodiester linkages of a primer sequence. While UNIT 15.1
describes in detail the optimization of standard PCR applications using unmodified
primers, this unit expands upon these concepts to describe the use of CleanAmp Primers
in a variety of PCR-based applications. Basic Protocol 1 covers the applications of stan-
dard thermal cycling PCR, fast thermal cycling PCR, and multiplex PCR, while Basic
Protocol 2 focuses on one-step reverse-transcription PCR (RT-PCR). While RT-PCR
is traditionally performed in two steps (see UNIT 15.5), this unit describes an efficient
one-step protocol that employs modified PCR primers. The protocols for each of these
applications can be adapted to be performed in real-time PCR (see Alternate Protocol),
allowing for quantification of nucleic acid targets.

CleanAmp is a registered trademark of TriLink Biotechnologies (http://www.

trilinkbiotech.com/).

A problem frequently encountered during PCR is the off-target amplification that results
from hybridization of primers to one another or to regions of the nucleic acid target with
lesser complementarity, and their consequent extension. These events occur during the
less stringent room temperature setup conditions under which thermostable DNA poly-
merases show a low level of activity. The undesired products not only reduce amplicon
yield, but also decrease specificity. To further improve PCR performance, a number of
approaches termed hot start have been developed. Typically, hot-start activation in PCR
The Polymerase
Chain Reaction

Current Protocols in Molecular Biology 15.9.1-15.9.15, October 2009 15.9.1

Published online October 2009 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471142727.mb1509s88 Supplement 88
Copyright C 2009 John Wiley & Sons, Inc.
 aims to block DNA polymerase extension at ambient temperatures, where nonspecific
product formation is more likely. Hot-start activation during the initial denaturation step
of PCR reverts the DNA polymerase back to its fully active form, allowing replication
to occur at the higher-stringency temperatures. The most commonly used approaches
to hot-start PCR result in the thermal activation of the DNA polymerase using methods
such as thermolabile antibodies (Mizuguchi et al., 1999; Eastlund and Mueller, 2001)
and chemical modifications (Birch et al., 1998; Moretti et al., 1998).

The protocols in this unit describe a method in which the primers, rather than the
polymerase, are activated by increased temperature. This primer-based hot-start PCR
approach uses thermolabile phosphotriester primer modifications that can easily be in-
troduced onto the 3 terminal phosphodiester linkages of standard primer sequences via
standard oligonucleotide protocols (Lebedev, 2009; Fig. 15.9.1). The presence of the
CleanAmp 4-oxo-tetradecyl (OXT) phosphotriester modifications prevents primer ex-
tension at the lower temperatures of PCR setup. A hot-start thermal activation step at
elevated temperatures removes the modification and generates the corresponding unmod-
ified primer, which supports amplification of the desired target (Fig. 15.9.2). CleanAmp
Primers present a hot-start activation mechanism that provides a controlled supply of
extendable primer during PCR cycling, which greatly improves amplification specificity.
This primer-based approach to hot-start PCR can be used with common thermostable
DNA polymerases, such as Taq DNA polymerase, without the need for highly modified

CleanAmp turbo primers CleanAmp precision primers

single 4-oxo-tetradecyl modification double 4-oxo-tetradecyl modification

Heat-Activatable
Primers for Hot Figure 15.9.1 Thermolabile 4-oxo-tetradecyl (OXT) primer modifications. X indicates the ther-
Start PCR molabile OXT modification group.

15.9.2
Supplement 88 Current Protocols in Molecular Biology
 5
3 5
3 5
CA 95 C 5
denaturation
3 5 72 C
5 extension
CA
5 48 - 60 C
annealing
3 5
5 3 Key:
25 C CA: single or double CleanAmp modification
setup conditions DNA polymerase

Figure 15.9.2 Proposed hot-start activation mechanism using CleanAmp Primers.

hot-start enzymes. Applications such as fast-cycling PCR, multiplex PCR, and one-step
RT-PCR aim to save time and obtain results faster; however, the specificity of the reaction
tends to be compromised. CleanAmp Primers show similar or improved benefit in these
advanced applications when compared to other hot-start technologies.

One particular application in which CleanAmp Primers excel is one-step RT-PCR.

Traditionally, RT-PCR experiments are performed in two steps, since one-step setups
are often plagued by decreased sensitivity that can result from undesired PCR primer
extension during the RT step. Hot-start approaches such as wax barriers have been used
to separate the reaction components so that only the necessary components are available
during the RT step, with the remainder of the components necessary for the PCR step
released into the reaction after an elevated-temperature incubation (Tanzer et al., 1999;
Sears and Khan, 2003; Fan et al., 2006). While effective, the use of barriers is not prac-
tical for use in one-step RT-PCR setups employing multiple samples, due to the large
number of manipulations. In contrast, the use of OXT-modified PCR primers greatly
reduces polymerase mispriming during the RT step and also provides the advantage of
using a standard, unmodified oligo(dT) or random primer in the RT step, rather than a
gene-specific primer. Furthermore, all reaction components can be added at the start of
the protocol, allowing for a more streamlined procedure with improved performance.
Although double OXT-modified (CleanAmp Precision) primers can be used in combi-
nation with hot-start polymerasebased polymerases (Basic Protocol 2) and also with
more thermostable RT polymerases such as SuperScript III RT (Invitrogen), there is no
need to invest in highly modified enzymes because the CleanAmp Primers provide good
results with less expensive, unmodified enzymes, such as Moloney Murine Leukemia
Virus Reverse Transcriptase (M-MLV RT) and Taq DNA polymerase.

STRATEGIC PLANNING
There are two basic protocols described in this unit. Basic Protocol 1 describes materials
and methods needed to perform a standard 3-step thermal cycling PCR protocol using
OXT-modified CleanAmp Primers. Within this protocol, there are two branch points to
describe how to adapt this protocol for fast thermal cycling PCR and multiplex PCR. Basic
Protocol 2 describes the use of CleanAmp Primers in one-step RT-PCR methodologies in
which the PCR primers are protected from extension during the RT step by the presence
of the OXT modification group. The ability to use temperature-dependent control of
primer utilization allows for improved specificity and for a completely unique approach
to RT-PCR setup.
The Polymerase
Chain Reaction

15.9.3
Current Protocols in Molecular Biology Supplement 88
 Choice of CleanAmp Primer type
There are two forms of CleanAmp Primers, differing from each other in the rate of
thermal activation to form the corresponding unmodified primers. At elevated temper-
atures (95 C), the single-OXT-modified CleanAmp Turbo Primers are activated more
quickly than the double-OXT-modified CleanAmp Precision Primers. For different PCR
applications and PCR performance needs, select one of the two varieties of CleanAmp
Primers:

CleanAmp Turbo Primers (Single-OXT-modified): Fast-cycling or two-step thermal cy-

cling protocols, where annealing and extension steps are combined; multiplexed PCR;
optimized protocols; improved amplicon yield.

CleanAmp Precision Primers (Double-OXT-modified): Standard, three-step thermal cy-

cling protocols; one-step reverse-transcription PCR (RT-PCR); improved specificity of
amplification and limit of detection; greatest reduction in mispriming/primer dimer for-
mation.

BASIC PRIMER-BASED HOT-START PCR USING STANDARD THERMAL

PROTOCOL 1 CYCLING
The following protocol is suggested for PCR amplification reactions that employ Taq
DNA polymerase in standard three-step thermal cycling protocols. Use singly modified
CleanAmp Turbo Primers to obtain a robust amplicon yield, or doubly modified Precision
Primers for the greatest reduction in off-target formation, such as mispriming. This
protocol can be modified into a fast thermal cycling protocol (step 6b) by combining the
annealing and extension steps of PCR into a single step, resulting in a two-step thermal
cycling protocol. In order to reduce the duration of PCR and obtain rapid, reliable results,
higher concentrations of PCR reagents (relative to standard PCR) are required. The
denaturation step is very short; therefore the faster-deprotecting CleanAmp Turbo Primers
are recommended. This protocol can also be modified to perform hot-start multiplex PCR.
Multiplex PCR reactions amplify multiple targets in a single reaction. Each multiplex
reaction requires multiple primer pairs, one per target of interest, increasing the chance
of off-target formation compared to standard PCR. The following protocol employs
CleanAmp Turbo Primers and Taq DNA polymerase in a PCR amplification reaction that
can be used to simultaneously detect up to nine targets (Shum and Paul, 2009). The use
of CleanAmp Turbo Primers allows for specific formation of only the intended targets,
while providing robust amplicon formation. This protocol differs from standard PCR in
that the concentration of dNTPs is doubled. As the number of targets in the multiplex
reaction increases, the units of DNA polymerase can be increased up to four-fold, with
a corresponding increase in magnesium chloride up to 4 mM final concentration. In
addition, for further optimization, KCl can be increased up to 100 mM final concentration.

Setting up the reaction

Thaw the CleanAmp Primer DMSO stock solution at room temperature. Vortex and
pulse centrifuge two to three times to thoroughly mix. Do not heat the stock solution
to thaw, as heating will result in removal of the CleanAmp Primer modification group.
CleanAmp Primers are provided in a DMSO solution at 200 M; if the setup requires
a lower concentration, remove a small aliquot of CleanAmp Primers and dilute with
either molecular biologygrade water or buffer (pH 7 to 9) until the desired working
concentration is attained. Since CleanAmp Primer modifications are more stable in
DMSO than in aqueous diluents, for maximal performance, dilutions should be used
Heat-Activatable immediately, with any remainder discarded.
Primers for Hot
Start PCR

15.9.4
Supplement 88 Current Protocols in Molecular Biology
Materials
10 PCR buffer: 200 mM TrisCl/500 mM KCl, pH 8.3 at 25 C (available in
prepared form from Invitrogen)
1 M KCl (for multiplex PCR; see Note below)
50 mM MgCl2 stock (Invitrogen)
10 mM dNTP mixture: 10 mM each of dATP, dCTP, dGTP, and dTTP
(New England Biolabs)
5 U/l Taq DNA polymerase (Invitrogen)
CleanAmp Primers (available made-to-order through custom oligonucleotide
synthesis service of TriLink BioTechnologies); alternatively, purchase
CleanAmp phosphoramidites (Glen Research or TriLink BioTechnologies) and
synthesize CleanAmp Primers in-house using solid-phase oligonucleotide
synthesis (see http://www.trilinkbiotech.com/cleanamp/Amidite procedure.pdf
and Lebedev, 2009)
Template DNA (10 pg/l to 1 g/l recommended concentration)
Thin-walled PCR tubes
Automated thermal cycler
UV transilluminator
Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A)
NOTE: In multiplex PCR, two or more primer pairs are introduced into the reaction.
Although the upper limit for how many CleanAmp Primer pairs can be introduced into a
multiplex reaction has yet to be determined, reactions containing up to nine primer pairs
have been demonstrated (Shum and Paul, 2009).

NOTE: When performing multiplex PCR, for optimal results, titrate KCl to between 70
and 100 mM final concentration (Henegariu et al., 1997), being sure to take into account
the different amounts of KCl that various PCR buffers contain (most, including the 1
concentration of the buffer described here, contain 50 mM KCl).

1. Prepare a reaction master mix according to the recipe provided in Table 15.9.1,
containing all components except for the DNA template sample. Add each of the
components as shown in Table 15.9.1 (multiply amounts by the number of reactions
needed plus one) into a microcentrifuge tube, on ice.
A slight excess of volume is thus prepared to compensate for pipetting losses.
For best results, add CleanAmp Primers at the end of the setup, since extended exposure
in the master mix may initiate their deprotection.

2. Mix the master mix gently by pipetting up and down. Pulse-spin in a microcentrifuge,
if necessary, to gather the solution at the bottom of the tube.
3. Aliquot 45 l of master mix per thin-walled PCR tube to be used in the experiment.
4. Add 5 l (containing 10 pg to 1 g) of template DNA to each reaction tube for a
final reaction volume of 50 l.
For different final reaction volumes, scale the reagent volumes in Table 15.9.1 up or down
proportionally and adjust the amount of master mix that will be added to each reaction,
ensuring that 1 to 5 l of template are added to the reaction.

5. Pulse-spin PCR tubes to remove bubbles and collect the reaction solution at the
bottom of the tube.

The Polymerase
Chain Reaction

15.9.5
Current Protocols in Molecular Biology Supplement 88
Table 15.9.1 Master Mix Reagents for Standard Thermal Cycling PCR, Fast Thermal Cycling PCR, and Multiplex PCR
for Reactions with a Final Volume of 50 l

Standard thermal cycling PCR Fast thermal cycling PCR Multiplex PCR
Final Volume for 1 Final Volume for 1 Final Volume for 1
Component
concentration reaction concentration reaction concentration reaction

10 PCR buffer 1 5 l 1 5 l 1 5 l
MgCl2 (50 mM) 2.5 mM 2.5 l 5.0 mM 5 l 2.5-4 mM Variable
dNTP mixture 0.2 mM 1 l 0.8 mM 4 l 0.4 mM 2 l
(10 mM of each)
DNA polymerase 0.025 U/l Variable 0.1-0.2 U/l Variable 0.05-0.1 U/l Variable
(Variable U/l)
CleanAmp Forward 0.1-0.5 Ma,b Variable 0.5-2 Ma,b Variable 0.1-0.5 Ma,b Variable
Primer
CleanAmp Reverse 0.1-0.5 Ma,b Variable 0.5-2 Ma,b Variable 0.1-0.5 Ma,b Variable
Primer
Sterile deionized water Up to 45 l Up to 45 l Up to 45 l
Total volume (l) 45 l 45 l 45 l
a In initial experiments, it is recommended to perform a titration of primer concentration for optimal performance. Note that the optimal concentration
for CleanAmp Primers is not necessarily the same as for unmodified primers and is often higher.
b Use CleanAmp Turbo and Precision Primers for standard thermal cycling PCR, and use CleanAmp Turbo Primers for fast thermal cycling and
multiplex PCR.

6. Place the tubes into a thermal cycler with a heated lid and:

a. For standard, three-step thermal cycling conditions and multiplex PCR, employ
the following thermal cycling protocol:

1 cycle: 2 to 10 min 95 C (initial dentaturation)

25 to 50 cycles: 30 sec 95 C (denaturation)
30 sec 48 to 65 C (annealing)
30 sec to 2 min 72 C (extension)
1 cycle: 10 min 72 C (final extension).

The annealing temperature should be chosen for optimal PCR performance. Most primer-
design software provides users with a recommended annealing temperature. In general, it
should be 5 C lower than the lowest primer melting temperature. Otherwise, the annealing
temperature can also be optimized experimentally either by using a thermal cycler with
gradient functionality or by performing several sequential experiments in which the
annealing temperature is varied. When some product other than the desired target is
formed, the annealing temperature should be increased 1 to 2 C.
b. For fast thermal cycling conditions, employ the following thermal cycling
protocol:

40 to 50 cycles: 10 sec 95 C (denaturation)

30 sec 62 to (annealing/extension).
72 C.

For fast thermal cycling, the annealing/extension temperature should be chosen by per-
forming the following calculation: annealing/extension temperature ( C) = (72 C +
Heat-Activatable average Tm of primers)/2.
Primers for Hot
Start PCR

15.9.6
Supplement 88 Current Protocols in Molecular Biology
 7. Analyze 20 l of reaction product by agarose gel electrophoresis (UNIT 2.5A) using
the appropriate percentage agarose gel, and ethidium bromide or another double-
stranded binding dye. Visualize the gel on a UV transilluminator.
When the PCR product needs to be conserved for subsequent use, dilute the entire PCR
product by adding an equivalent volume of water and load 20 l on an electrophoresis
gel.
This step may be omitted if performing a real-time experiment (see Alternate Protocol).

PRIMER-BASED HOT-START ONE-STEP REVERSE-TRANSCRIPTION PCR BASIC

PROTOCOL 2
Reverse transcription PCR (RT-PCR) differs from standard PCR in that it uses RNA,
such as total RNA or poly(A) RNA, as template instead of DNA. RT-PCR consists of
two sequential protocols that involve a lower-temperature reverse transcription (RT) step
using a poly(dT), random, or gene-specific primer, followed by an elevated-temperature
PCR step. Performing both steps in a one-step RT-PCR protocol reduces the number
of manipulations but results in a higher probability for off-target amplification. While
the use of gene-specific primer improves the specificity of one-step RT-PCR reactions
(Wacker and Godard, 2005), the use of an unmodified primer for the RT step [poly(dT)
or random primer] and the doubly modified Precision CleanAmp Primers for the PCR
amplification step reduces the probability of mispriming events typically caused during
one-step reaction setups. This allows the user the flexibility to employ an RT primer
of choice, such as a poly(dT), random, or gene-specific primer. There are advantages
and disadvantages to using one- versus two-step PCR. One-step RT-PCR does not allow
a common cDNA to be prepared and does not allow for optimization of the RT step,
whereas two-step does. One-step RT-PCR is recommended for use when there are many
samples to run, and two-step RT-PCR is recommended when the RNA sample is limiting
and optimization is needed. The following protocol is suggested for one-step RT-PCR
and multiplex one-step RT-PCR amplification reactions that employ the commonly used
Taq DNA polymerase and Moloney Murine Leukemia Virus reverse transcriptase (M-
MLV-RT) enzymes.

Materials
10 PCR buffer: 200 mM TrisCl/500 mM KCl, pH 8.3 at 25 C (available in
prepared form from Invitrogen)
1 M KCl (for multiplex PCR; see Note: below)
50 mM MgCl2 stock (Invitrogen)
10 mM dNTP mixture: 10 mM each of dATP, dCTP, dGTP, and dTTP (New
England Biolabs)
50 M standard poly(dT) primer or random primer
40 U/l RNase inhibitor (Ambion)
200 U/l M-MLV Reverse Transcriptase (Invitrogen) or 200 U/l SuperScript III
Reverse Transcriptase (Invitrogen)
5 U/l Taq DNA polymerase (Invitrogen)
CleanAmp Precision Primers (TriLink BioTechnologies, Inc.): forward and reverse
Template: 0.1 to 1 g/g total RNA or poly(A) RNA sample
Thin-walled PCR tubes
Automated thermal cycler
UV transilluminator
Additional reagents and equipment for RT-PCR (UNIT 15.5) and agarose gel
electrophoresis (UNIT 2.5A)

The Polymerase
Chain Reaction

15.9.7
Current Protocols in Molecular Biology Supplement 88
 Table 15.9.2 Master Mix Reagents for One-Step RT-PCR for Reactions with a Final
Volume of 50 l

Component Final concentration Volume for 1 reaction

10 PCR buffer 1 5 l
Magnesium chloride (50 mM) 1.5 mM 1.5 l
dNTP mixture (10 mM of each) 0.16 mM 0.8 l
Poly(dT) or random primer (50 M) 1 M 1 l
RNase inhibitor (Variable U/l) 0.1 U/l Variable
a
Reverse transcriptase (Variable U/l) 1 U/l Variable
DNA polymerase (Variable U/l) 0.0125 U/l Variable
b
CleanAmp Precision Forward Primer 0.1-0.5 M Variable
b
CleanAmp Precision Reverse Primer 0.1-0.5 M Variable
Sterile deionized water Up to 49 l
Total volume (l) 49 l
a M-MLV-RT or SuperScript III RT. If other enzyme is used, follow the manufacturers recommendations.
b In initial experiments, it is recommended to perform a titration of primer concentration for optimal perfor-
mance. Note that the optimal concentration for CleanAmp Primers is not necessarily the same as for unmodified
primers and is often higher.

NOTE: When adapting this protocol from single-plex to multiplex one-step RT-PCR, the
units of Taq DNA polymerase can be increased up to four times to increase amplicon
yield without any additional changes to the reaction setup. Using the buffer conditions
described in Table 15.9.2, there is no need to increase the MgCl2 concentration when the
units of Taq are quadrupled. When increasing the number of targets to more than three,
the use of a reverse transcriptase that is active at higher temperatures, such as SuperScript
III RT, can improve the specificity of the reaction.

1. Prepare a master mix according to the recipe provided in Table 15.9.2 containing all
components except for the RNA template sample. Add each of the components as
shown in Table 15.9.2 (multiply amounts by the number of reactions needed plus
one) into a microcentrifuge tube, on ice.
A slight excess of volume is thus prepared to compensate for pipetting losses.
For best results, add CleanAmp Primers at the end of the setup, since extended exposure
in the master mix may initiate their deprotection.

2. Mix the master mix by gently pipetting up and down. Pulse-spin in a microcentrifuge
if necessary to collect the solution at the bottom of the tube.
3. Aliquot 49 l of master mix per thin-walled PCR tube to be used in the experiment.
4. Add 1 l (containing 0.1 to 1 g) of total RNA or poly(A) RNA to each reaction
tube for a final reaction volume of 50 l.
For different final reaction volumes, the reagent volumes in Table 15.9.2 up or down
proportionally and adjust the amount of master mix that will be added to each reaction,
ensuring that 0.5 to 1 l of RNA template are added to the reaction.
Place the PCR tubes on ice until they are transferred to the thermal cycler to prevent RNA
degradation.

5. Pulse-spin PCR tubes to remove bubbles and collect the reaction solution at the
Heat-Activatable
Primers for Hot bottom of the tube.
Start PCR

15.9.8
Supplement 88 Current Protocols in Molecular Biology
 6. Place the tubes into a thermal cycler with a heated lid and cycle using the following
standard RT-PCR thermal cycling conditions (also see UNIT 15.5):

1 cycle: 30 min 42 C (reverse transcription)

1 cycle: 10 min 95 C (initial denaturation)
30 to 40 cycles: 30 sec 95 C (denaturation)
30 sec 48 to 60 C (annealing)
30 sec 72 C (extension)
1 cycle: 5 min 72 C (final extension).

The final 5-min extension cycle may be omitted if performing a real-time experiment.
When using SuperScript III RT, increase the reverse transcription temperature from 42 C
to 55 C.

7. Analyze 20 l of reaction product by agarose gel electrophoresis (UNIT 2.5A) using

the appropriate percentage agarose gel, and ethidium bromide or another double-
stranded binding dye. Visualize the gel on a UV transilluminator.
When the PCR product needs to be conserved for subsequent use, dilute the entire PCR
product by adding an equivalent volume of water and load 20 l on an electrophoresis gel.
This step may be omitted if performing a real-time experiment (see Alternate Protocol).

PRIMER-BASED HOT-START PCR WITH REAL-TIME DETECTION ALTERNATE

PROTOCOL
Each of the previously described protocols can be adapted for real-time PCR to detect
and quantify the amplification of a targeted nucleic acid after each cycle. This proto-
col describes the procedures for two commonly used detection methods. SYBR Green
detection uses a fluorescent dye that intercalates with the double-stranded DNA. Taq-
Man Probe detection uses a dual-labeled hydrolysis probe that fluoresces after target
hybridization and subsequent 5 exonuclease cleavage of the probe (UNIT 15.8). The main
difference between these two detection methods is that SYBR Green will bind to and
detect any double-stranded DNA, including the nonspecific reaction products, whereas
TaqMan Probe provides an extra level of specificity by hybridization only to comple-
mentary nucleic acid sequences. Other quantification methods include molecular beacon
probes and scorpion probes, which are mentioned in UNIT 15.8 and are further described
in http://dyes.gene-quantification.info/.

Additional Materials (also see Basic Protocol 1)

For SYBR Green detection:
10,000 SYBR Green I nucleic acid stain (Invitrogen)
Passive reference: 1 mM ROX dye (Stratagene)
For TaqMan Probe detection:
TaqMan Probe (see UNIT 15.8 for probe design)
Passive reference: 1 mM ROX dye
Thermal cycler capable of real-time detection
Follow the desired basic protocol described above, applying the following alterations
depending on the type of detection desired:

1. Add to the master mix ROX and either SYBR Green or TaqMan Probe, making the
necessary adjustments to the water volume in the master mix preparation.

a. For SYBR Greenbased detection, include 30 or 300 nM passive ROX refer-

ence dye (refer to the on real-time instrument manufacturer for recommended
concentration) and 0.15 SYBR Green I nucleic acid stain in the reaction. The Polymerase
Chain Reaction

15.9.9
Current Protocols in Molecular Biology Supplement 88
 b. For TaqMan Probebased detection, include 30 or 300 nM passive ROX reference
dye (refer to the real-time instrument manufacturer for recommended concentra-
tion) and 50 to 200 nM TaqMan Probe in the reaction.
The optimal TaqMan Probe concentration should be determined by performing serial
dilutions and identifying the concentration that provides the earliest Cq and maximal
fluorescence intensity (UNIT 15.8). A typical probe concentration is 0.1 M.
IMPORTANT NOTE: For the stock solutions and dilutions of the passive ROX reference
dye and TaqMan Probe, it is very important to keep the tubes protected from light at all
times (i.e., wrap tubes with aluminum foil). The dilution of passive ROX reference dye can
be stored at 4 C for 1 month if kept in the dark.

2. Incubate the reactions in a thermal cycler capable of real-time detection, and collect
fluorescence data at the completion of the annealing step of each cycle. The final
extension step may be omitted. In SYBR Green experiments, following the PCR
cycle, append the manufacturers recommended thermal cycling conditions for the
melting curve analysis to the end of your protocol; a typical melting curve thermal
cycling protocol is:

1 cycle: 95 C for 1 min (denaturation)

1 cycle: 55 C for 30 sec (annealing)
1 cycle: 95 C for 30 sec (melting).

3. Optional: Perform agarose gel electrophoresis (UNIT 2.5A).

The agarose gel electrophoresis analysis step can often be omitted since the amplification
curve will yield product formation data, especially after the assay is well developed.
Typically agarose gel analyses are performed in conjunction with real-time data to confirm
a positive result in an unknown sample or to investigate an atypical result.

COMMENTARY
Background Information itation (Barnes and Rowlyk, 2002). While
the wax-barrier technique requires the most
Hot-start PCR approaches hands-on time, it has shown utility in one-step
Since the inception of hot-start PCR, the
RT-PCR protocols as a result of the physical
development of new PCR-based applications
separation of the DNA polymerase and PCR
has multiplied. Technologies that employ hot-
primers from the RT portion of the reaction.
start PCR are common in molecular diagnos-
Other hot-start approaches are polymerase-
tics where fast, accurate results are necessary.
based, and include noncovalent binding to the
The hot-start mechanism is based upon block-
polymerase by antibodies (Mizuguchi et al.,
ing, in some fashion, the activity of the DNA
1999; Eastlund and Mueller, 2001), chemi-
polymerase at ambient temperatures to pre-
cal modifications to the polymerase (Birch
vent nonspecific extension of polymerase sub-
et al., 1998; Moretti et al., 1998), and the
strates, and then releasing or activating the
use of accessory proteins (Borns, 2007; Kubu
polymerase after a short, high-temperature in-
et al., 2008). The use of antibodies and ac-
cubation (Chou et al., 1992). As a result, the
cessory proteins allows for fast dissociation,
amplification is more specific and efficient, re-
with rapid release of viable DNA polymerase
sulting in a cleaner and robust PCR product
into the reaction. On the other hand, chem-
(UNIT 15.1).
ically modified DNA polymerases, such as
Over the years, several hot-start activa-
AmpliTaq Gold, may require longer activation
tion techniques have been developed, with
times, but the slower release of active en-
the methods that have been commercialized
zyme, referred to as time-release, can pro-
described in Table 15.9.3. Early approaches
vide additional specificity. Moreover, not ev-
to hot-start activation employed separation
ery commercially available DNA polymerase
of reagent components using wax barriers
has been converted to the corresponding mod-
Heat-Activatable
(Chou et al., 1992; Tanzer et al., 1999), en-
ified hot-start DNA polymerase, limiting users
Primers for Hot capsulated DNA polymerase (Setterquist and
Start PCR
to a much smaller subset of DNA polymerases
Smith, 1996), and magnesium ion precip-
15.9.10
Supplement 88 Current Protocols in Molecular Biology
for their experiments. Much like the chemi-
cally modified DNA polymerases, chemically
modified primers (Kaboev et al., 2000; Ullman
et al., 2002; Ankenbauer et al., 2003; Bonner,
2003; Laird and Niemiec, 2004) and chem-
ically modified dNTPs (Koukhareva et al.,
2008) present a hot-start activation mecha-
nism in which a heat-activation step provides
a controlled release of unmodified primers
and dNTPs, respectively, into the reaction. For
each of these last two types of hot-start ap-
proaches, users can employ their DNA poly-
merase of choice, using modified primers or
modified dNTPs to convert the DNA poly-
merase into its hot-start version.
The added price per reaction for a variety
of hot-start approaches has been calculated,
and is included on Table 15.9.3. In each case,
the price is estimated based on the assumption
that the end user will employ standard reaction
conditions50-l reaction with 0.125 U of
DNA polymerase, 10 pmol of forward and re-
verse primer, 0.2 mM dNTPs, with 0.4 mM for
modified (CleanAmp) dNTPs. However, be-
cause certain PCR protocols may require more
enzyme, more dNTPs, or more primer than the
standard reaction conditions, the price per re-
action will vary depending on the complexity
of the downstream reaction. For example, in
multiplexed PCR, the price per reaction em-
ploying CleanAmp Primers will be multiplied
by the number of primer pairs in the reaction.
Overall, with the number of permutations that
are possible with a standard PCR protocol, the
listed price points are for a specific set of con-
ditions and can, as a result, vary significantly.
Heat-activatable primers for hot-start PCR
The approach that is described in this unit is
based on temperature-sensitive chemical mod-
ifications to the phosphodiester backbone of
the primers (Lebedev et al., 2008; Lebedev,
2009). While primer-based hot-start PCR tech-
niques that employ CleanAmp Primers are
easy to implement into a number of PCR-based
applications; they excel in more advanced
applications such as fast PCR, multiplex PCR,
and one-step RT-PCR. In fast-cycling pro-
tocols, CleanAmp Primers are employed at
higher concentrations, with the need for up to
a 5-fold excess to allow for sufficient unmodi-
fied primer release during the cycling. While,
as a general rule, higher primer concentrations
increase the probability of off-target amplicon
formation, the presence of the OXT group al-
lows for a much higher primer concentration
to be used without the complication of off-
target amplicon formation. In multiplex PCR,
Current Protocols in Molecular Biology
typical problems include nonspecific priming
and unequal amplification of certain targets
(termed PCR selection and PCR drift; Wag-
ner et al., 1994). By using CleanAmp Turbo
Primers, a controlled release of viable primer
is introduced into the reaction, resulting in
greater target amplicon yield and more uni-
form amplification of each target, with mini-
mal optimization in primer concentration. The
biggest advantage of CleanAmp Primers is in
one-step RT-PCR. Although performing both
the RT and PCR reactions in the same tube as
a one-step RT-PCR protocol may reduce setup
time, a one-step reaction tends to be less sen-
sitive than a two-step reaction, even with the
use of a gene-specific primer (GSP) for robust
RT performance (Wacker and Godard, 2005).
However, by using an unmodified RT primer
and CleanAmp PCR primers, the reaction sen-
sitivity is increased without the need to use a
GSP. Moreover, by performing RT-PCR in one
step and using unmodified poly(dT) primers
(UNIT 15.6) or random primers instead of a GSP,
the cDNA synthesis step can be reproduced
for each reaction, allowing for a copy of the
total RNA to be made each time a reaction
is set up. Repetition of the RT step can help
to eliminate copy-number bias that may result
from a faulty cDNA synthesis reaction or from
preferential amplification of specific regions
of the total RNA when more than one GSP is
used. Furthermore, when multiplexing, multi-
ple GSPs would be needed, and several factors,
including RNA secondary structure and prim-
ing efficiencies, may interfere with the GSP
extension. In contrast, by using a single RT
primer, the cDNA product formation will be
less biased, allowing for better quantification
of the RNA in a given sample. Overall, the use
of primers modified with thermolabile modifi-
cations provides an alternative mechanism of
action in hot-start PCR and shows great poten-
tial for improved PCR performance.
Critical Parameters
Storage and handling of the CleanAmp
Primers
Storage and handling of the CleanAmp
Primers play an important role in obtaining
good results. CleanAmp Primers should be
stored at 20 C or cooler to avoid loss of the
4-oxo-tetradecyl (OXT) modification group.
The stock solution should be thawed for 5 to
15 min at room temperature. Under no circum-
stances should the stock solution be heated to
thaw it, as heating will result in removal of the The Polymerase
CleanAmp Primer modification group. Prior to Chain Reaction
15.9.11
Supplement 88
Table 15.9.3 Comparison of Different Approaches to Hot Start Activation in PCR

Hot-start Advantages (Adv)/ Price increase/

Commercial name Mechanism
approach disadvantages (Dis) reactiona,b

Physical AmpliWax Wax beads separate key (Adv) Allows for a single tube $$$$
barriers PCRGems (Applied PCR components (reaction one-step RT-PCR setup in which the
Biosystems) buffer, DNA polymerase, PCR primers and DNA polymerase
and template) prior to are kept separate from the reverse
heating transcription reagents.
(Dis) Requires an extra manipulation
step to form the wax barrier, which
can be problematic, especially for
more high-throughput setups.
TaqBead Hot Start Wax bead encapsulates the (Adv) Beads contain 1.25 U of Taq, $$$
DNA polymerase DNA polymerase until allowing for precise control of DNA
(Promega) release at elevated polymerase addition to a setup.
temperatures (Dis) Beads need to be added
manually introducing potential
sample contamination.
(Dis) Compartmentalized DNA
polymerase limits the flexibility to
vary polymerase units in a set-up
beyond 1.25 U increments.
Polymerase- Platinum Taq Antibodies bind to the (Adv) High-fidelity amplifications of $$$
based Hot (Invitrogen enzyme until they long targets. Improved specificity
Start Corporation) dissociate at higher and yield. Reduced initial hot start
approach temperatures activation (only 2 min).
(Dis) Requires optimization.
AmpliTaq Gold Chemical modification to (Adv) Chemical hot-start capability $$
(Applied Biosystems) the lysines of DNA can release active enzyme in a
polymerase; modifications time-release manner.
are released at high (Dis) Requires a heat activation step
temperatures of 1-10 min at 95 C.
Accessory Hot Start-IT DNA binding protein (Adv) Provides high specificity in $$
proteins (USB/Affymetrix) sequesters primers at low multiplex reactions. It is portable to a
temperatures variety of thermostable polymerases.
Paq5000 Hotstart Protein binds to primed (Adv) High sensitivity and robust $
DNA Polymerase templates and prevents yield.
(Stratagene/Agilent) extension until inactivation (Adv) Extension time can be
at elevated temperatures shortened to perform a faster PCR.
Essential Rockstart PCR buffer Reagent precipitates (Dis) Requires incubation of reagents $$
magnesium (DNA Polymerase essential magnesium ion for a few minutes to form the
ion precipi- Technology) until thermal cycling when magnesium precipitate; not suited for
tation the magnesium dissolves high-throughput setups.
Modified CleanAmp Primers One or two temperature (Adv) Allow for selective primer $
primers (TriLink sensitive phosphotriester activation in sequential enzymatic
Bio-Technologies, modification groups to the reactions, such as one-step RT-PCR.
Inc.) 3 end of the primer which (Dis) Requires a 2-10 min initial
dissociate at high denaturation at 95 C.
temperatures
continued
Heat-Activatable
Primers for Hot
Start PCR

15.9.12
Supplement 88 Current Protocols in Molecular Biology
Table 15.9.3 Comparison of Different Approaches to Hot Start Activation in PCR, continued
Hot-start
Commercial name Mechanism
approach
Modified CleanAmp dNTP Thermolabile modification (Adv) Compatible with a variety of
dNTPs Mix (TriLink Bio group to the 3 hydroxyl of DNA polymerases, allowing
-Technologies, Inc.) the dNTPs
a Values for the price increase per reaction were determined for a 50-l reaction which included 0.125 U of DNA polymerase, 10 pmol of forward
and reverse primer, 0.2 mM dNTPs, with 0.4 mM CleanAmp dNTPs.
b $:Up to $0.25; $$: $0.26-0.5; $$$: $0.51-1; $$$$: $1 and up.
use, the stock should be vortexed two to three
times to mix well. For maximal performance,
divide the stock solution into several tubes of
smaller volumes upon receipt to prevent con-
tamination and extended room temperature ex-
posure over many uses. After removing the de-
sired aliquot for the PCR reaction, the unused
portion should be returned to the freezer as
quickly as possible. DMSO provides the stock
solution with more stability than water. Dilu-
tions of the stock should be made using water
or aqueous buffer (pH 7 to 9) as the diluent, and
stored on ice. The diluted solution should be
used immediately, and any remainder should
be discarded.
Initial denaturation time
For adequate CleanAmp Primer deprotec-
tion, an initial denaturation time of 2 to 10 min
at 95 C is recommended for standard ther-
mal cycling protocols. If using a chemically
modified DNA polymerase, such as AmpliTaq
Gold DNA polymerase, in conjunction with
CleanAmp Primers, an initial denaturation of
10 to 20 min may be required. For the modi-
fied conditions of fast cycling, shorter denat-
uration times at 94 C can be employed (see
Troubleshooting below for further details).
The total thermal cycling time can be
reduced by decreasing the initial denatura-
tion time and by increasing the CleanAmp
Primer concentration. When adding an excess
of CleanAmp Primers, there will be more un-
protected primer available for the polymerase
after the denaturation period.
Choice of DNA polymerase
CleanAmp Primers can be employed with
a variety of commercially available DNA
polymerases. The compatibility of CleanAmp
Current Protocols in Molecular Biology
Advantages (Adv)/ Price increase/
disadvantages (Dis) reactiona,b
$$
conversion of a standard DNA
polymerase into a hot-start version.
(Dis) Requires a heat activation step
of 1-10 min at 95 C.
Primers with a number of thermostable
DNA polymerases increases PCR specificity
without the need for a hot-start enzyme.
Table 15.9.4 lists DNA polymerases and ven-
dors that have been validated for their utility
with CleanAmp Primers.
The described protocols can be adapted
for use of different DNA polymerases with
the following alterations to the protocol. For
reactions containing Pfu DNA polymerase
(exo+ and exo), DyNAzyme DNA poly-
merase (New England Biolabs), Tth DNA
polymerase, Tfi DNA polymerase, and Deep
VentR DNA polymerase (New England Bio-
labs), each polymerase should be used with the
corresponding buffers provided by the manu-
facturer. For each DNA polymerase, the stan-
dard thermal cycling reaction conditions are
identical to those for Taq DNA polymerase
(Basic Protocol 1), with the exception of Deep
VentR DNA polymerase, which employs dou-
ble the number of units of DNA polymerase.
Use of CleanAmp Primers with
commercially available PCR master mixes
It is not recommended to use CleanAmp
Primers with commercially available PCR
master mixes, as the additives in these mixes
have been found to slow the rate of release
of the CleanAmp OXT modification group
(unpub. observ.). In many cases, this de-
creased rate of release results in PCR inhi-
bition. Should it be necessary to use commer-
cially available master mixes, improved per-
formance may be obtained by the use of higher
primer concentrations or longer initial denat-
uration times. See Basic Protocols 1 and 2 for
recommended usage of CleanAmp Primers,
and Tables 15.9.1 and 15.9.2 for master mix
recipes that do not cause the abovementioned
inhibition.
The Polymerase
Chain Reaction
15.9.13
Supplement 88
 Troubleshooting
Several parameters can be optimized de-
pending on the source of the problem. A few
troubleshooting suggestions are described be-
low for situations where amplicon yield is low
or absent and for situations where nonspecific
product formation dominates. For further trou-
bleshooting, refer to the appropriate literature
from the polymerase manufacturer.
No amplification product or low amplicon
yield
1. Poor or nonexistent PCR product forma-
tion is often a result of insufficient activation
of CleanAmp Primers during thermal cycling.
Ways to solve it are:
a. Increase the concentration of CleanAmp
Primers to up to 2 M. Primer/template sys-
tems that are prone to mispriming often re-
quire a much lower primer concentration
than systems that are prone to primer dimer
formation.
b. Optimize the duration of the initial de-
naturation time to up to 20 min when used in
conjunction with chemically modified DNA
polymerases or commercially available PCR
master mixes.
c. Verify that the level of DMSO that was
introduced into the reaction by the CleanAmp
Primers is not inhibitory to the reaction. Typ-
ically, between 0% and 10% DMSO can be
employed in PCR; however, in some instances,
DMSO may cause PCR inhibition. Therefore,
only add DMSO as a reaction supplement if
absolutely necessary and to a maximum of
2% (v/v).
d. If a commercially available PCR mas-
ter mix and its recommended guidelines are
being employed, attempt experiments follow-
ing the appropriate setup from the appropriate
protocol in this unit instead.
Table 15.9.4 DNA Polymerase Vendors Validated for Their Utility with CleanAmp
Primers
DNA polymerase name
Taq DNA polymerase (native and recombinant)
Taq DNA polymerase (recombinant)
EconoTaq DNA polymerase
Pfu DNA polymerase (exo+ and exo)
DyNAzyme DNA polymerase
Tth DNA polymerase
Heat-Activatable Tfi DNA polymerase
Primers for Hot
Start PCR Deep VentR DNA polymerase
15.9.14
Supplement 88
2. Poor PCR performance may also result
from an unoptimized thermal cycling protocol.
Ways to optimize the thermal cycling protocol
include:
a. Increase extension time. Generally ex-
tension times should be 1 to 2 min per kb of
target.
b. Increase the number of thermal cycles in
5-cycle increments.
c. Optimize annealing temperature.
3. The cause of the low or no amplifica-
tion may be due to a problem with reagents or
reaction conditions:
a. Prepare fresh reagents, including reaction
buffer and dNTPs.
b. Verify that the template is of good quality
and of sufficient quantity.
c. Verify primer design to ensure adequate
complementarity to the DNA target.
d. Optimize the MgCl2 concentration.
Nonspecific product formation
1. To reduce mis-priming and other off-
target primer extensions:
a. Titrate the concentration of CleanAmp
Primers to as low as 50 nM.
b. Reduce extension time to avoid spurious
amplification products.
c. Reduce the amount of DNA polymerase.
d. Titrate the amount of template DNA.
e. Optimize the MgCl2 concentration.
Time Considerations
Preparing, cycling, and visualizing results
of the PCR reactions in an electrophoresis gel
can be completed in one day. For all proto-
cols, setting up the reaction requires 30 min.
The PCR amplification time varies depend-
ing on the thermal cycling conditions. Prepar-
ing the gel and visualizing the samples takes
2 hr.
Vendor
Invitrogen
New England Biolabs
Lucigen
Stratagene/Agilent
Finnzymes
USB
Invitrogen
New England Biolabs
Current Protocols in Molecular Biology
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The Polymerase
Chain Reaction
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Supplement 88