CHAPTER TWENTY TWO
Explanatory Chapter:
Troubleshooting PCR
Kirstie Canene-Adams1
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
1
Corresponding author: e-mail address: kirstieadams26@[Link]
Contents
1. Theory 271
2. Equipment 272
3. Materials 272
4. Protocol 272
References 278
Abstract
The purpose of this chapter is to assist with any problems you are having with the poly-
merase chain reaction (PCR) protocol from General PCR.
1. THEORY
The simple theory behind PCR is to use custom-designed primers,
template DNA, a thermostable DNA polymerase, and nucleotides (dNTPs)
to amplify a particular region of DNA so that it can be easily detected or
manipulated. There are many applications for PCR including gene cloning,
manipulation, mutagenesis, genotyping, detection and diagnosis of patho-
gens, DNA sequencing, and analyzing expression levels of a gene.
PCR comprises three main steps:
• Denaturation: this separates the double-stranded DNA into single-
stranded DNA.
• Annealing: the primers bind to the complementary target sequence in the
template DNA.
• Extension: the thermostable DNA polymerase synthesizes the comple-
mentary DNA strand, starting from the 30 -end of the primer.
Methods in Enzymology, Volume 529 # 2013 Elsevier Inc. 271
ISSN 0076-6879 All rights reserved.
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272 Kirstie Canene-Adams
All PCR should be evaluated to confirm that a PCR product of the expected
size was made. This is most commonly done by agarose gel electrophoresis
(see Agarose Gel Electrophoresis). This chapter assists in the troubleshooting
of PCR in the event that there was no product produced or a product of the
wrong size was produced.
2. EQUIPMENT
PCR thermal cycler
Micropipettors
Aerosol barrier pipet tips
Sterile microcentrifuge tubes
0.2-ml thin-walled PCR reaction tubes or plates
Surface decontamination wipe (e.g., DNA AWAY®, Molecular
Bioproducts)
Agarose gel electrophoresis equipment
3. MATERIALS
Template DNA
Primers
dNTPs
Taq DNA polymerase (or other thermostable DNA polymerase)
10 PCR buffer
Magnesium chloride (MgCl2, 25 mM)
Sterile ultra pure water
Ethidium bromide
Normal melting agarose
TAE buffer
DNA ladder
4. PROTOCOL
Rather than being a standard protocol, this chapter lists common
problems with PCR, the reasons why they might have occurred, and steps
that can be taken to prevent these problems in the future. Most often, after
performing PCR, an agarose gel is run with the PCR product alongside a
DNA ladder to verify the size of the product. Two problems can be imme-
diately apparent: first, there is no PCR product, and second, there is a PCR
Explanatory Chapter: Troubleshooting PCR 273
Table 22.1 Troubleshooting the lack of a PCR product seen on an agarose gel
PCR
problem Possible reasons Fixes
No • Problem with reagents • Repeat the experiment, as a reagent
product may have been unintentionally
formed left out
• Check that the reagents had been fully
thawed and mixed thoroughly
• Try a new vial of dNTPs as they can
be damaged by repeated freeze–
thaw cycles
• Use a different polymerase
• Try using an additive such as DMSO
or glycerol
• Quality of the template • Use a NanoDrop spectrophotometer
DNA is poor to check the quantity and quality of
template DNA
• Too much or too little • Remake template DNA. Older stocks
starting template can degrade, particularly for
genomic DNA
• Set up a series of reactions with
varying amounts of template (between
10 and 200 ng DNA)
• Primers are • Check that the primers have been
not working diluted to the correct concentration
• Make sure that the sequence of the
primer is what you expected it to be
synthesized as. Typos are common!
• Increase primer concentration
• Redesign the primers
• PCR inhibitors are • Demonstrate that a control gene or
present in the another DNA sequence can be effec-
template DNA tively amplified using the same tem-
plate DNA. If it can, there is another
problem with the PCR reaction
• Problem with the set- • Check the cycler – Are the tempera-
tings on thermal cycler tures and times as you expect?
• Change the annealing temperature.
Find the optimum temperature by
using a gradient cycler and test a range
from the lowest primer Tm to 10 C
below the Tm
Continued
274 Kirstie Canene-Adams
Table 22.1 Troubleshooting the lack of a PCR product seen on an agarose gel—cont'd
PCR
problem Possible reasons Fixes
• Try the reaction in another cycler –
the calibration of the one you are
using may be off
• The template DNA is • Use a polymerase buffer intended for
GC-rich GC-rich templates. Test a full range of
the additive (e.g., GC melt)
Table 22.2 Troubleshooting PCR products which are either too long or too short
PCR problem Possible reasons Fixes
Wrong sized • Cross-contamination • Use the following order
product formed when setting up the reac-
tions: actual samples, posi-
tive controls, and then
negative controls
• One or both primers • Use aerosol barrier
anneal to an off-target site pipettor tips
on the template • Pre- and postamplification
samples should be
manipulated in physically
separated areas. Set up a
separate lab bench area and
a set of pipettors for
running gels
Long nonspecific • One or both primers • Decrease the annealing
products anneal to an off-target site and/or extension time
on the template • Decrease the extension
temperature to 62–68 C
• Increase the
annealing temperature.
• Increase the MgCl2 con-
centration to 3–4.5 mM,
while keeping the dNTP
concentration constant
• Use less primers, DNA
template, and/
or polymerase
• If none of the above works,
BLAST the primer for
repetitive sequences and
try designing new
primer(s)
Explanatory Chapter: Troubleshooting PCR 275
Table 22.2 Troubleshooting PCR products which are either too long or too short—
cont'd
PCR problem Possible reasons Fixes
Short nonspecific • Small PCR bands about • Increase the annealing
products or the size of one primer or temperature and/or time.
‘primer dimers’ both together, which are Try to find the optimal
formed by the annealing of annealing temperature
a primer to itself or to the using a gradient
other primer PCR machine
• One or both primers • Increase the extension time
anneal to an off-target site and/or temperature to
on the template, resulting 74–78 C
in a very short • Titrate the MgCl2 con-
PCR product centration to 3–4.5 mM,
while keeping the dNTP
concentration constant
• Use less primer or
Taq polymerase
• Try a ‘hot start’ polymerase
instead of a
standard polymerase
• Increase the amount of the
DNA template
• If none of the above works,
BLAST the primer for
repetitive sequences and
try designing new
primer(s)
product(s) but it is the wrong size. Table 22.1 gives some reasons and pos-
sible fixes for when there is no PCR product produced. Table 22.2 gives
some reasons and possible fixes for when there is a PCR product, but it
is the wrong size.
Since there are five main reagents in a PCR, you can alter any of them in
order to optimize the reaction for your particular gene of interest and prod-
uct. It is preferred to only change one aspect of the reaction at a time. The
following gives examples of how each reagent, MgCl2, Taq DNA polymer-
ase, the primers, dNTPs, and template DNA, can cause problems in PCR
and acceptable ranges of concentrations for each reagent.
MgCl2: Magnesium plays several roles in PCR: it is an essential cofactor
for thermostable DNA polymerases, it stabilizes double-stranded DNA, and
it elevates the Tm. A deficient or low Mg2þ concentration requires stricter
base pairing when the primers and DNA anneal, and can cause a small or no
276 Kirstie Canene-Adams
yield of PCR product. If Mg2þ ions are in excess, there can be an increased
yield of nonspecific products, and the reliability of DNA polymerases can be
reduced, promoting the misincorporation of nucleotides and the samples
appearing as a smear on the gel. Since dNTPs sequester Mg2þ ions, any
alteration in dNTP concentrations will require additional changes in the
concentration of MgCl2. Normally, the MgCl2 concentration is best
between 1 and 4 mM.
Taq DNA polymerase: Low levels of Taq polymerase can cause incom-
plete primer elongation or untimely termination of the PCR product syn-
thesis during the extension step. Too much polymerase will result in an
excessive background of unwanted DNA fragments, which will look like
a smear on the gel. A markedly overabundant amount of polymerase can
cause the reaction to completely fail with no product formed at all.
A polymerase concentration of 1 unit per 25 ml of reaction mix is sufficient
to generate a clean PCR product.
Primers: Low primer concentration generally results in a cleaner product,
yet raising the primer concentration does not lead to a corresponding
increase in the amount of product formed. Using elevated concentrations
of primers can result in the creation of primer-dimers, nonspecific primer
binding, and the generation of undesirable PCR products. Conversely, to
amplify short target sequences, such as 100 base pairs, a higher number of
molecules of PCR product are required to provide a specified amount of
amplified DNA (in nanograms). In this case, a higher primer concentration
could be beneficial. The suggested primer concentration is between 0.1 and
1 mM of each primer. Primer base composition and priming sites can also
dramatically affect the PCR performance, see chapter Explanatory chapter:
PCR -Primer design.
dNTPs: A suboptimal concentration of nucleotides can lead to incom-
plete primer elongation or untimely termination of synthesis at some point
in the extension step. Excessive dNTP concentrations can inhibit PCR,
preventing product formation. The usual dNTP concentration is between
40 and 200 mM for each of the four dNTPs. To amplify longer DNA frag-
ments, a higher dNTP concentration may be required.
DNA template: The concentration of the template DNA should be
balanced with the number of cycles in the reaction. When the total amount
of DNA is extremely small, there is increased likelihood of loss, contaminat-
ing DNA from impurities, and/or degradation. Using too much DNA
can lead to off-target annealing of the primers as well as poor DNA synthesis
Explanatory Chapter: Troubleshooting PCR 277
due to the obstructed diffusion of polymerase. However, reducing
the number of cycles may help to resolve these problems. Contamination
can come from unlikely sources such as the dust floating in the air or particles
of skin or hair from your body, which can carry both DNA and DNA-
degrading nucleases. To prevent this, autoclave the tubes, wear gloves,
and clean the working space using an oxidizing substance (e.g., 6%
H2O2), 100% ethanol, or a surface decontamination wipe (e.g., DNA
AWAY®, Molecular Bioproducts).
Weak amplification of your target can be improved by:
• Increasing the amount of primers, DNA template, and/or polymerase.
• Checking primer sequences for mismatches and/or increasing the primer
length by five nucleotides (see also Explanatory chapter: PCR -Primer
design).
• Increasing the annealing time or decreasing the annealing temperature.
• Increasing the number of cycles.
• Trying an additive of 5% (v/v, final concentration) DMSO or glycerol.
• Extracting the weak PCR product from an agarose gel using a DNA gel
purification kit and using this as the template in a new reaction.
Nonspecific band amplification can be fixed by:
• Repeating the reaction with a negative control, such as water. The non-
specific bands are most likely from contaminating foreign DNA. If this is
the problem, use new stocks for all reagents. Also, remember to always
use autoclaved PCR tubes and wear gloves.
• Using less DNA template.
• Increasing the annealing time if the nonspecific products are shorter than
your target. If they are longer than your target, reduce the
annealing time.
• Increasing the annealing temperature.
• Redesigning the primer(s) to increase the length at its 30 -end as the extra
bands may be from similar sequences to your target. Increasing the 30
primer length by adding further matching base pairs will make the primer
more specific for your target because correct sequence binding will be
enhanced while the extension of the nonspecific sequences will be
blocked. When trying to eliminate aberrant bands, it is usually not a good
idea to increase the length of a primer at its 50 -end, because this will
increase its ability to anneal to the off-target sequences. In some cases
shorter primers will work better because they do not base pair as well
to the off-target sequences.
278 Kirstie Canene-Adams
REFERENCES
Related Literature
Thermo Scientific “PCR Troubleshooting Guide” Assessed May–June 2010.
[Link]
Bitesize Bio Beta, Brain Food for Scientists “The Essential PCR Troubleshooting Checklist”
Nick Oswald, PhD; Assessed May–June 2010.
[Link]
“PCR Troubleshooting: The Essential Guide” Michael L. Altshuler; Caister Academic Press;
March 2006, Review online and Assessed May–June 2010.
[Link]
Referenced Protocols in Methods Navigator
General PCR
Agarose Gel Electrophoresis
Explanatory chapter: PCR -Primer design