What do you do?

Haemorrhagic Fever Viruses

I am a biomedical scientist in the Department of Veterinary Microbiology, University of Ibadan, Nigeria.

I am involved in bacterial and viral research of Medical and Veterinary importance. These include
bacteria responsible for sexually transmitted diseases among others. I also work Astroviruses,
Haemorrhagic Fever viruses which includes Ebola, Yellow fever, Dengue, Westnile virus and Lassa
Fever infections. I am involved the laboratory diagnosis of haemorrhagic fever viruses/ Arboviruses and
parasite vectors such as cullicoides which transmits Blue Tongue virus. The diagnosis is done by
carrying out screening of serological clinical samples by ELISA. This is followed by molecular diagnosis
through the extraction of viral genomes, mix preparation and amplification by polymerase chain reaction
(PCR). I detect the amplicons by Gel electrophoresis and observed using Biorad Gel Doc imager. I carry
out further characterization of the pathogen through genomic sequencing and phylogenetic analysis after
Blasting with MEGA software and then linkage with Los Alamos geneBank.

I also conduct practical for undergraduate and postgraduate medical, dental and veterinary medicine
students in veterinary and medical virology, principles and application of PCR, ELISA, Western blotting
and Fluorescence microscopy. As a researcher and diagnostician, I have been working on the isolation
and molecular characterization and genetic diversity of respiratory viruses in children with acute lower
respiratory infection in southwestern Nigeria including vaccine development.

Benefits to me, community and Institution

My attendance in this conference will offer me a source of motivation as a young scientist. It will afford
me the opportunity to learn how other scientist solved their problem which may be similar to mine. This
can help me move past the roadblock in my research. It will give me the opportunity to make contact and
network with other researchers and also share my own views. This contact may result in collaborations
for me in future. To my institution; since my utmost goal is in the area of research and teaching, this
conference will benefit my institution (Faculty of Veterinary Medicine) University of Ibadan such that the
knowledge gained will be imparted on Undergraduate and Postgraduate students and also on my
professional colleagues.

My community will also benefit greatly in the areas of diseases prevention. Also my community will
benefit from awareness on the mode of transmission and control sexually transmitted infections especially
those relating to emerging and re-emerging human infections and methods of prevention through
education and proper management of infected persons.

Working with PCR

 General Considerations
o Setting up the laboratory to avoid contamination
o Special equipment
 o PCR reaction components
o Thermal cycling profile for standard PCR
o Properties of PCR enzymes

 Factors to Consider in RT-PCR

o Choice of RT-PCR enzymes
o Choice of reverse transcription primers
o Preparation of template RNA
o Primer design
o RT-PCR procedures

 Prevention of Carryover Contamination

o Preventing carryover contamination with uracil-DNA glycosylase
o Preventing carryover contamination in RT-PCR

 Troubleshooting
o No product
o Misincorporation or low fidelity
o Nonspecific bands
o Smeared bands
o Low yield

General Considerations
Setting up the laboratory to avoid contamination
The ability of the polymerase chain reaction to amplify a single molecule means that trace amounts of DNA
contaminants could serve as templates, resulting in amplification of the wrong template (false positives).
Consider the points mentioned below to avoid PCR contamination from sources such as:

 Laboratory benches, equipment, and pipetting devices, which can be contaminated by previous DNA
preparations, by plasmid DNA, or by purified restriction fragments.
 Cross-contamination between samples.
 Products from previous PCR amplifications.

Laboratory facilities

 At a minimum, use physically separated working places for:

o template preparation before PCR
o setting up PCR reactions
 o post-PCR analysis
 Use thin-walled PCR tubes which are DNase- and RNase-free.
 Use special aerosol-resistant pipette tips and a dedicated (used only for PCR) set of pipettes,
preferably positive displacement pipettes.
 If possible, set up PCR reactions under a fume hood that is equipped with UV light. Store a
microcentrifuge and disposable gloves which are used only for PCR under the fume hood.

Sample handling

 Use sterile techniques and always wear fresh gloves when working in the PCR area. Change gloves
frequently, especially if you suspect they have become contaminated with solutions containing
template DNA.
 Always use new and/or sterilized glassware, plasticware, and pipettes to prepare PCR reagents and
template DNA.
 Autoclave all reagents and solutions that can be autoclaved without affecting their performance. Of
course, primers, dNTPs, and Taq DNA Polymerase should not be autoclaved. Have your own set of
PCR reagents and solutions that are used only for PCR. Store these reagents in small aliquots.
 When pipetting DNA, avoid creating aerosols that could carry contaminants.
 Always include control reactions, for example a negative ("no DNA") control which contains all reaction
components except the template DNA, and a positive control that has been successfully used in
previous PCRs.

Special equipment
Type of thermal cycler
A thermal cycler must, at a minimum, accurately and reproducibly maintain the three PCR incubation
temperatures (see Thermal Cycling Profile for Standard PCR), change from one temperature to another
("ramp") over a definable time, arrive at the selected temperatures without significant over- or undershoot, and
cycle between the temperatures repeatedly and reproducibly.
Note: Cycling conditions have to be adjusted depending on the respective thermal cycler or primer/template
combinations.
Type of reaction tubes
The reaction tubes affect the rate at which heat transfers from the thermal cycler to the reaction mixture.
Therefore, preferably use thin-walled reaction tubes that are designed for PCR and that fit precisely into the
wells of the particular brand of thermal cycler you are using.

PCR reaction components

Template
The quality of the template influences the outcome of the PCR. For instance, large amounts of RNA in a DNA
template can chelate Mg2+ and reduce the yield of the PCR. Also, impure templates may contain polymerase
inhibitors that decrease the efficiency of the reaction.
Note: To get the purest template, always use a purification product specifically designed to purify DNA, such
as the High Pure PCR Template Preparation Kit.
The integrity of the template is also important. Template DNA should be of high molecular weight. To check
the size and quality of the DNA, run an aliquot on an agarose gel. When testing a new template, always
include a positive control with primers that amplify a product of known size and produce a good yield.
The amount of template in a reaction strongly influences performance in PCR. The recommended amount of
template for standard PCR is:

 A maximum of 500 ng of human genomic DNA

 1 – 10 ng bacterial DNA
 0.1 – 1 ng plasmid DNA

Low amounts of template, for example, <10 ng human genomic DNA, will require specific reaction
modifications, such as changes in cycle number, redesign of primers, use of Hot Start, etc.
Primers
In most PCR applications, it is the sequence and the concentration of the primers that determine the overall
assay success. For convenience, several primer design software programs are available. These can be used
to ensure that the primer sequences have the following general characteristics:

 18 – 24 bases long
 No internal secondary structure
 40 – 60% G/C
 Balanced distribution of G/C and A/T rich domains.
 Not complementary to each other at the 3´ ends (primer-dimers will not form).
 Melting temperature (Tm) that allows annealing temperatures of +55 to +65°C (for maximum specificity
use temperatures of +62 to +65°C).

Note: Optimal annealing temperatures are often higher than the Tm of the primers (approximately +5 to
+10°C) and have to be determined empirically. For both primers, the Tm should be similar.
Bases that do not hybridize to the template may be added at the 5´ end of a primer (e.g., for introducing
restriction sites into the amplification product). Primer concentrations between 0.1 and 0.6 µM are generally
optimal. Higher primer concentrations may promote mispriming and accumulation of nonspecific product.
Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of
desired product.
Note: For some systems, a higher primer concentration (up to 1 µM) may improve results. When testing new
primers, always include a positive control reaction with a template that has been tested for function in PCR.
This control shows whether the primers are working. The Human Genomic DNA from Roche is a good control
template for evaluation of human primer sequences.
Choice of DNA polymerase
The choice of a DNA polymerase can profoundly affect the outcome of PCR. For most routine PCR, Taq DNA
Polymerase has long been the standard PCR enzyme. However, Taq DNA Polymerase has its limitations. For
most assays, the optimum amount of thermostable DNA polymerase (or a blend of polymerases) will be
between 0.5 and 2.5 units / 50 µl reaction volume. Increased enzyme concentrations sometimes lead to
decreased specificity.
MgCl2 concentration
Mg2+ forms soluble complexes with dNTPs to produce the actual substrate that the polymerase recognizes.
The concentration of free Mg2+depends on the concentrations of compounds that bind the ion, including dNTP,
free pyrophosphate (PPi) and EDTA. For best results, always determine the optimal Mg 2+ concentration
empirically. The optimal Mg2+ concentration may vary from approximately 1 - 5 mM. The most commonly used
Mg2+ concentration is 1.5 mM (with dNTPs at a concentration of 200 µM each). Mg2+ influences enzyme activity
and increases the Tm of double-stranded DNA. Excess Mg 2+ in the reaction can increase nonspecific primer
binding and increase the nonspecific background of the reaction.
Note: For easy optimization of Mg2+, see the PCR Optimization Kit from Roche.
Deoxynucleotide triphosphate (dNTP) concentration
Always use balanced solutions of all four dNTPs to minimize polymerase error rate. Imbalanced dNTP
mixtures will reduce Taq DNA Polymerase fidelity.
Note: For maximum convenience, a premixed, balanced mixture of dNTPs such as the PCR Nucleotide
Mix may be added to the reaction mixture as a single reagent. In addition, PCR
grade dATP, dGTP, dCTP, dTTP and a Deoxynucleoside Triphosphate Set, PCR Grade are available.
If you increase the concentration of dNTPs, you must also increase the Mg 2+ concentration. Increases in dNTP
concentration reduce free Mg2+, thus interfering with polymerase activity and decreasing primer annealing. For
prevention of carryover contamination, a higher concentration of dUTP is usually used in place of dTTP (for
details, see Preventing carryover contamination with uracil-DNA glycosylase). The final dNTP concentration
should be 50 – 500 µM (each dNTP). The most commonly used dNTP concentration is 200 µM.
pH
Generally, the pH of the reaction buffer supplied with the corresponding thermostable DNA polymerase (pH
8.3 – 9.0) will give optimal results. However, for some systems, raising the pH may stabilize the template and
enhance results.
Note: Determining the optimal reaction pH can be simplified with the PCR Optimization Kit from Roche.
Reaction additives
In some cases, adding the following compounds can enhance the efficiency or specificity of PCR:

 Betaine (0.5 – 2 M)
 Bovine serum albumin (BSA; 100 ng/50 µl)
 Detergents
 Dimethylsulfoxide (DMSO; 2 – 10%) (v/v)
 Gelatine
 Glycerol (1 – 5%) (v/v)
 Pyrophosphatase (0.001 – 0.1 units/reaction)
 Spermidine
 T4 Gene 32 protein
 Formamide

The effect of these additives must be determined empirically, such as with the PCR Optimization Kit from
Roche.

Thermal cycling profile for standard PCR

Initial denaturation
It is essential to denature the template DNA completely. Initial heating of the PCR mixture for 2 minutes at +94
to +95°C is enough to completely denature complex genomic DNA so that the primers can anneal to the
template as the reaction mix is cooled. If the template DNA is only partially denatured, it will tend to "snap-
back" very quickly, preventing efficient primer annealing and extension, or leading to "self-priming", which can
lead to false-positive results.
Denaturation step during cycling
Denaturation at +94 to +95°C for 20 – 30 seconds is usually sufficient, but this must be adapted for the
thermal cycler and tubes being used. For example, longer times are required for denaturation in 500 µl tubes
than in 200 µl tubes. If the denaturation temperature is too low, the incompletely melted DNA "snap-backs" as
described earlier, thus giving no access to the primers. Use a longer denaturation time or higher denaturing
temperature for GC-rich template DNA.
Note: Never use a longer denaturation time than absolutely required for complete denaturation of template
DNA. Unnecessarily long denaturation times decrease the activity of Taq DNA Polymerase.
Primer annealing
For most purposes, annealing temperature has to be optimized empirically. The choice of the primer annealing
temperature is probably the most critical factor in designing a high specificity PCR. If the temperature is too
high, no annealing occurs, but if it is too low, non-specific annealing will increase dramatically. Primer-dimers
will form if the primers have one or more complementary bases so that base pairing between the 3´ ends of
the two primers can occur.
Primer extension
For fragments up to 3 kb, primer extension is normally carried out at +72°C. Taq DNA Polymerase can add
approximately 60 bases per second at +72°C. A 45-second extension is sufficient for fragments up to 1 kb. For
extension of fragments up to 3 kb, allow about 45 seconds per kb. However, these times may need to be
adjusted for specific templates. For improved yield, use the cycle extension feature of the thermal cycler. For
instance, perform the first 10 cycles at a constant extension time (e.g., 45 seconds for a 1 kb product). Then,
for the next 20 cycles, increase the extension time by 2–5 seconds per cycle (e.g., 50 seconds for cycle 11, 55
seconds for cycle 12, etc.). Cycle extension allows the enzyme more time to do its job, because as PCR
progresses, there is more template to amplify and less enzyme (due to denaturation during the prolonged high
PCR temperatures) to do the extension.
Cycle number
In an optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product
that is easily detectable on a gel stained with ethidium bromide. Most PCRs should, therefore, include only 25
to 35 cycles. As cycle number increases, nonspecific products can accumulate (see Figure 1).

Figure 1: Effect of excessive cycling on impure and pure templates. A PCR product (245 bp amplicon
from exon 6 of the dopamine 2 receptor gene) was reamplified in a series of reactions. In one set of
experiments, the template was not purified before it was used. In the second set, the template was purified by
agarose gel electrophoresis before reamplification. In both sets, the template was amplified for either 40, 60,
or 72 cycles. Aliquots (8 µl) of the products were analyzed on a 3% agarose gel.
MWM: Molecular Weight Marker
40, 60, and 72: Number of amplification cycles.
Result: In both sets, the lowest number of cycles (40) produced the most specific product. In both the 60 and
72 cycle amplifications, a smear appeared which contained multimeric "specific" PCR products.
Photo courtesy of U. Finckh and A. Rolfs, Free University of Berlin, Germany.
Final extension
Usually, after the last cycle, the reaction tubes are held at +72°C for 5 – 15 minutes to promote completion of
partial extension products and annealing of single-stranded complementary products.

Properties of PCR enzymes

Table 1 shows the application profile of PCR enzymes provided by Roche.

Table 1: Application profile of PCR enzymes provided by Roche.

Length The length of the sequences which can be amplified.

The extent of nonspecific amplification in particular, if difficult templates are to be amplified. The higher the spe
Specificity
lesser by-products are generated.

Reproducibility Consistency of results obtained with different enzyme lots.

Dependence of amplification efficiency on the amount of template. If the template is limited, an enzyme system
Sensitivity
sensitivity should be used.

Vulnerability of an enzyme system to contaminating agents or other factors which reduce the amplification effic
Robustness
as strong secondary structures.
 Error rate of an enzyme system as defined by the relative number of misincorporated nucleotides compared to
Accuracy
Polymerase.

Carryover Defines whether an enzyme system can be used with dUTP instead of dTTP to apply procedures for PCR carry
Prevention prevention using uracil DNA glycosylase (UNG).

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Factors to Consider in RT-PCR
Choice of RT-PCR enzymes
Obviously, in RT-PCR, a major factor to consider is the choice of reverse transcriptase used to synthesize
cDNA. Since each of the available enzymes has different enzymatic properties, one may be more suitable for
a specific experiment than the others. The most important enzyme properties are discussed below.
Temperature optima
Higher incubation temperatures can help eliminate problems of template secondary structure. In addition, high
temperature improves the specificity of reverse transcription by decreasing false priming. Thus, thermoactive
reverse transcriptases that can be incubated at high temperatures (+50 to 70°C) are more likely to produce
accurate copies of mRNA, especially if the template has a high GC content.
Note: At these high temperatures, use only specific primers; do not use oligo(dT) or random hexamer primers.
Divalent ion requirement
Most reverse transcriptases require a divalent ion for activity. Enzymes that use Mg 2+ are likely to produce
more accurate cDNA copies than those that use Mn2+, since Mn2+ adversely affects the fidelity of DNA
synthesis.
Specificity and sensitivity
Reverse transcriptases have differing ability to copy small amounts of template (sensitivity). They also differ in
their ability to transcribe RNA secondary structures accurately (specificity).
Enzyme profiles
Table 2 shows the application profiles of RT-PCR enzymes and kits provided by Roche.
Table 2: Application profiles of RT-PCR enzymes and kits provided by Roche.
 

Choice of reverse transcription primers

Priming affects the size and specificity of the cDNA produced. There are three types of primers that may be
used for reverse transcription:

 Oligo(dT)12–18, which binds to the endogenous poly(A)+ tail at the 3´ end of mammalian mRNA. This
primer often produces a full-length cDNA.
 Random hexanucleotides, which bind to mRNA at a variety of complementary sites and lead to
partial length (short) cDNAs. Random hexanucleotides may be ideal for overcoming the difficulties
presented by extensive secondary structure in the template. These primers may also transcribe more
efficiently 5´ regions of the mRNA.
 Specific oligonucleotide primers, which selectively prime the mRNA of interest. This type of primer
has been used very successfully in diagnostic assays.

Preparation of template RNA

Successful RT-PCR requires a high quality, intact RNA template. Use the following guidelines to help prepare
this template:

 To minimize the activity of RNases that are released during cell lysis, include Protector RNase
Inhibitor in the lysis mix or use methods that simultaneously disrupt cells and inactivate RNases.
  Take steps to eliminate all potential sources of RNase contamination from glassware, plasticware,
reagents, etc.
 Use a product specifically designed for nucleic acid purification to prepare starting template RNA, such
as the RNA preparation products provided by Roche.
 Use purified mRNA as template, rather than total RNA. Starting with poly(A)+ mRNA will greatly
increase the likelihood of successful amplification of rare mRNAs, since the proportion of mRNA in a
total RNA preparation is quite low (typically, 1 – 5% of total RNA from a mammalian cell).
 If using mRNA as template, check the integrity of the mRNA by gel electrophoresis before using it in
RT-PCR. The mRNA should appear as a smear between approximately 500 bp and 8 kb. Most of the
mRNA should be between 1.5 kb and 2 kb.

Primer design
RT-PCR amplification of a particular mRNA sequence requires two PCR primers that are specific for that
mRNA sequence. The primer design should also allow differentiation between the amplified product of cDNA
and an amplified product derived from contaminating genomic DNA. There are two approaches to designing
the required primers (see Figure 2).

Figure 2: Primer design approaches.

Panel 1: Make primers that anneal to sequences in exons on both sides of an intron. With these primers, any
product amplified from genomic DNA will be much larger than a product amplified from intronless mRNA.
Panel 2: Make primers that span exon/exon boundaries on the mRNA. Such primers should not amplify
genomic DNA.
 

RT-PCR procedures
RT-PCR can be performed as either a two-step or an one-step procedure. Each has certain advantages.
1. Two-step procedures
A. Two tube, two-step procedure
In the first tube, first-strand cDNA synthesis is performed under optimal conditions, using either random
hexamers, oligo(dT) primers (generating a cDNA pool), or sequence-specific primers. An aliquot of the RT
reaction is then transferred to another tube (containing thermostable DNA polymerase, DNA polymerase
buffer, and PCR primers) for PCR.
Advantages of this approach: This method is useful for experiments where multiple transcripts have to be
analyzed from the same RT reaction or for specific applications, such as Differential Display Reverse
Transcription (DDRT) or Rapid Amplification of cDNA Ends (RACE). Also, since the RT reaction is performed
under optimal conditions, this approach produces the longest RT-PCR products (up to 14 kb in length, if the
appropriate enzymes are used).
B. One tube, two-step procedure
In the first step, reverse transcriptase produces first-strand cDNA in the presence of Mg 2+ ions, high
concentrations of dNTPs, and either specific or nonspecific [oligo(dT)] primers (reaction volume, 20 µl).
Following the RT reaction, an optimized PCR buffer (without Mg 2+ ions), a thermostable DNA polymerase, and
specific primers are added to the tube and PCR is performed. This approach may be useful when template
amounts are limited, since the entire RT reaction is used in the subsequent PCR.
A two-step procedure has the following advantages:

 Optimizes reaction conditions. The two-step format allows both reverse transcription and PCR to be
performed under optimal conditions to ensure efficient and accurate amplification.
 Provides flexibility. Two-step procedures allow the product of a single cDNA synthesis reaction to be
used for analysis of multiple transcripts. This flexibility is valuable for such specialized applications as
rapid amplification of cDNA ends (RACE) and Differential Display Reverse Transcription (DDRT).
 Amplifies long sequences. With the right combination of reverse transcriptase and thermostable
DNA polymerase, two-step RT-PCR can amplify RNA sequences up to 14 kb long.

2. One tube, one-step procedure (coupled RT-PCR)

Both cDNA synthesis and PCR amplification are performed with the same buffer and site-specific primers,
eliminating the need to open the reaction tube between the RT and PCR steps. In addition to the higher
sensitivity of this approach (as in the one tube, two-step reaction above), the one-step approach minimizes the
chance of contamination, since the entire reaction is performed with minimal pipetting steps and without
opening the tube. In addition, this approach permits direct analysis of a specific transcript, since the primers
used in both steps are sequence-specific. Finally, the thermoactive reverse transcriptase used in this
procedure allow a high RT reaction temperature (+50 to +72°C), which reduces false priming and increases
the specificity of the reaction by eliminating secondary mRNA structure.
A one-step procedure has the following advantages:

 Minimizes time required. The one-step reaction has fewer pipetting steps than the two-step reaction,
significantly reducing the time needed to perform RT-PCR and eliminating pipetting errors.
  Reduces the risk of contamination. The entire one-step reaction takes place in a single tube, with no
transfers required and no need to open the reaction tube (steps where contamination of the PCR
sample can take place).
 Improves the sensitivity and specificity of cDNA synthesis. Two characteristics of the one-step
reaction provide increased yield and efficiency: (1) the cDNA reaction is performed at a high
temperature (to eliminate problems with RNA secondary structure), and (2) the entire cDNA sample is
used as template for PCR.

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Prevention of Carryover Contamination
Preventing carryover contamination with uracil-DNA glycosylase
The polymerase chain reaction (PCR) can amplify a single molecule over a billionfold. Thus, even minuscule
amounts of a contaminant can be amplified and lead to a false-positive result. Such contaminants are often
products from previous PCR amplifications (carryover contamination). Therefore, researchers have developed
methods to avoid such contamination.
One common strategy is substituting dUTP for dTTP during PCR amplification, to produce uracil-containing
DNA (U-DNA). Treating subsequent PCR reaction mixtures with Uracil-DNA Glycosylase (UNG) prior to PCR
amplification and subsequent cleavage of apyrimidinic polynucleotides at elevated temperature (+95°C) under
alkaline conditions (during the initial denaturation step) will remove contaminating U-DNA from the sample
(see Figure 3). This method, of course, requires that all PCR reactions in the lab be carried out with dUTP
instead of dTTP.

Figure 3: Removal of contaminating U-DNA with uracil-DNA glycosylase.

Note the following when using dU-containing PCR products in downstream applications:

 PCR products containing dU perform as well as those containing dT when used as hybridization
targets or as templates for dideoxy sequencing.
  PCR products containing dU can be cloned directly, if they are transformed into ung–bacterial hosts.
 A dU-containing substrate is readily digested by some common restriction enzymes (e.g., Eco RI and
Bam HI), while others show reduced activity (e.g., Hpa I, Hind II, Hind III) on these substrates.
 We do not recommend the use of dU-containing DNA for protein binding or DNA-protein interaction
studies.

Preventing carryover contamination in RT-PCR

There are two ways of preventing carryover contamination when amplifying RNA:

 Use a one-tube, one-step RT-PCR procedure to minimize the number of handling and pipetting steps
and the number of times that the reaction tube must be opened. This minimizes the possibility of
carryover contamination in the RT-PCR itself.
 Perform one-step RT-PCR in the presence of dUTP, so that all products will contain dU and can be
removed from subsequent PCR with UNG (see Preventing Carryover Contamination with Uracil-DNA
Glycosylase). This prevents carryover contamination in subsequent PCRs.

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Troubleshooting
Here are some troubleshooting hints that we have gathered regarding PCR, based on the five most common
symptoms observed. Before you begin, please make sure that you have reviewed the other application hints.

No product
Here are some troubleshooting hints that we have gathered if your PCR yields no product.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. The amount of template in the reaction is not optimal
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human
DNA (105 - 106 copies) per 100 µl reaction. The amount of enzyme should be optimized for each template.
Suggestion:

 Titrate the amount of template in the reaction.

 Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range
(0.5 to 5.0 units).

3. An enzyme inhibitor is present in the reaction

Known inhibitors of PCR include:

 50 mM ammonium chloride
 EDTA (metal chelator)
 >0.8 µM hematin
  PBS (phosphate will bind free magnesium)
 >0.02 % sarcosyl
 0.5 M urea
 >5% DMF
 >10% formamide
 Heparin
 >20% PEG deoxycholate
 >0.01% SDS (can be reversed with equal molar ratio of NP40 and Tween 20)
 >10% DMSO
 >20% glycerol
 >0.4% N-octylglucoside
 Residual phenol
 >0.06% sodium deoxycholate
 Pyrophosphate

Suggestion: Reduce or remove the concentration of any inhibitor in the reaction mixture.

4. Primer annealing temperature is too high or too low
Primer annealing temperature is typically +50 to +60°C (may be higher or lower based on primer sequence
and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases 
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
5. Primers are degraded or not optimal
Primers should have the same number A and T's versus G and C's, and they should be at least 14 bases for
specificity.
Suggestions:

 If primers are short and A-T rich, add 0.9 - 2.0% (v/v) DMSO.
 If primers are G-C rich, add 1 - 10% (v/v) formamide.
 Double-check priming sequence, use primer design program if available.
 Check aliquot of primers on a gel to ensure they are not degraded.

6. Incomplete template denaturation

Insufficient heating during the denaturation step is a common cause of failure in a PCR reaction. It is very
important that the reaction reaches a temperature at which complete strand separation occurs. A temperature
of about +94°C for 2 minutes should be adequate in most cases.
As soon as the sample reaches +94°C, it can be cooled to the annealing temperature. Extensive denaturation
is probably unnecessary and limited exposure to elevated temperatures helps maintain maximum polymerase
activity throughout the reaction.
DNA reaction buffers with higher Mg concentration (4 - 5 mM) may require a higher denaturation temperature
to allow complete separation of the DNA template strands. It is recommended to use the supplied buffer
without adding further magnesium.
Suggestions:

 Increase initial denaturation temperature to +95 to +97°C.

 Denature DNA minus enzyme and buffer for 4 - 6 minutes.
 Increase cycling denaturation time 15 - 30 seconds.
 Try Hot Start technique.
 Closed circular DNA should be cleaved before the PCR, uncut circles renaturate too fast.

7. Machine-based error
Suggestions:

 Calibrate the heating block and confirm actual block temperature.

 Run a diagnostic program for the particular machine - contact the machine manufacturer for specifics.

8. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends

of primers
Suggestions:

 Increase initial denaturation temperature to +95 to +97°C.

 Denature DNA minus enzyme and buffer for 4 - 6 minutes.
 Increase cycling denaturation time 15 - 30 seconds.
 Try Hot Start technique.
 Add T4 Gene 32 protein 3 - 5 µl / ml.
 When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a
primer design program if available.
 Consider addition of cosolvent to reaction buffer:
o 3 - 15% DMSO
o 1 - 10% formamide
o 5 - 15% polyethylene glycol
o 10 - 15% glycerol

9. NaCl concentration above 50 mM

Suggestions: Reduce NaCl concentration
10. KCl concentration above 50 mM
Suggestions: Reduce KCl concentration
 
Misincorporation or low fidelity
Here are some troubleshooting hints that we have gathered regarding misincorporation or low fidelity of PCR
reactions.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:

 Check the concentration of stock solutions of all nucleotides.

 Double-check the final concentrations of all nucleotides.

3. The pH of the reaction buffer is too high

A pH of 8.3 is optimal.
Suggestions:

 pH of reaction buffer can be lowered to 5 - 6.

 Base substitution changes decreased by sixtyfold and frame-shift changes decreased by elevenfold
with a 3 unit pH decrease.

4. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends

of primers
Suggestions:

 Increase initial denaturation temperature to +95 to +97°C.

 Denature DNA minus enzyme and buffer for 4 - 6 minutes.
 Increase cycling denaturation time 15 - 30 seconds.
 Try Hot Start technique.
 Add T4 Gene 32 protein 3 - 5 µl / ml.
 When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a
primer design program if available.
 Consider addition of cosolvent to reaction buffer:
o 3 - 15% DMSO
o 1 - 10% formamide
o 5 - 15% polyethylene glycol
o 10 - 15% glycerol

5. Damaged template DNA

Suggestions:
  Minimize cycle number, since repeated heating/cooling at high pH can damage template (most
templates require 25 - 35 cycles).
 Check integrity of template DNA.
 Reduce cycling denature time to 15 - 30 seconds

Nonspecific bands
Here are some troubleshooting hints that we have gathered regarding nonspecific bands after PCR reactions.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:

 Check the concentration of stock solutions of all nucleotides.

 Double-check the final concentrations of all nucleotides.

3. DNA contamination/carryover
Suggestions:

 Test for carryover by performing PCR without adding target DNA.

 Avoid carryover (see below).

To prevent carryover, use good laboratory practices:

 Physically isolate PCR preps and products

 Autoclave solutions, tips, and tubes
 Aliquot reagents to minimize repeated sampling (no more than 20 reactions per aliquot)
 Eliminate aerosols by using positive-displacement pipettes
 Premix reagents
 Add DNA to reaction last
 Choose positive and negative controls carefully
 Soak gel box and combs in 1 M HCl to depurinate DNA
 Use new razor blades to excise bands
 Cover UV box with fresh plastic wrap
 Always use oil overlay

To eliminate contamination/carryover:
  UV irradiation: Mix all components, except template DNA; irradiate in clear 0.5 ml polypropylene tubes
in direct contact with glass transilluminator (254 and 300 nm UV bulbs) for 5 minutes.
 UNG digestion: Incorporate dUTP nucleotides into reaction and do subsequent uracyl DNA
glycosylase digestion.

4. Primer annealing temperature is too low

Primer annealing temperature is typically +50 to +60°C (may be higher or lower based on primer sequence
and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases 
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
5. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends
of primers
Suggestions:

 Increase initial denaturation temperature to +95 to +97°C.

 Denature DNA minus enzyme and buffer for 4 - 6 minutes.
 Increase cycling denaturation time 15 - 30 seconds.
 Try Hot Start technique.
 Add T4 Gene 32 protein 3 - 5 µl / ml.
 When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a
primer design program if available.
 Consider addition of cosolvent to reaction buffer:
o 3 - 15% DMSO
o 1 - 10% formamide
o 5 - 15% polyethylene glycol
o 10 - 15% glycerol

6. Primers are degraded or not optimal

Primers should have the same number A and T's versus G and C's, and they should be at least 14 bases for
specificity.
Suggestions:

 If primers are short and A-T rich, add 0.9 - 2.0% (v/v) DMSO.
 If primers are G-C rich, add 1 - 10% (v/v) formamide.
 Double-check priming sequence, use primer design program if available.
  Check aliquot of primers on a gel to ensure they are not degraded.

7. Primer concentration is too high

Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal).
8. Cycle number is too high
Most templates require 25 - 30 cycles.
Suggestion: Cycle number should be based on starting concentration of template DNA.
If the number of target molecules in Then we recommend the following
your sample is... number of cycles...
3 x 10 5
25 - 30
1.5 x 10 4
30 - 35
1.0 x 10 3
35 - 40
50 40 - 45
9. Incorrect template to enzyme ratio
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human
DNA (105 - 106 copies) per 100 µl reaction. The amount of enzyme should be optimized for each template.
Suggestions:

 Titrate the amount of template in the reaction.

 Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range
(0.5 to 5.0 units).

 
Smeared bands
Here are some troubleshooting hints that we have gathered regarding smeared bands after PCR reactions.
1. Non-optimal Mg2+ concentration
Suggestion: Titrate magnesium concentration using our PCR Optimization Kit.
2. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:

 Check the concentration of stock solutions of all nucleotides.

 Double-check the final concentrations of all nucleotides.

3. DNA contamination/carryover
Suggestions:

 Test for carryover by performing PCR without adding target DNA.

 Avoid carryover (see below).
To prevent carryover, use good laboratory practices:

 Physically isolate PCR preps and products

 Autoclave solutions, tips, and tubes
 Aliquot reagents to minimize repeated sampling (no more than 20 reactions per aliquot)
 Eliminate aerosols by using positive-displacement pipettes
 Premix reagents
 Add DNA to reaction last
 Choose positive and negative controls carefully
 Soak gel box and combs in 1 M HCl to depurinate DNA
 Use new razor blades to excise bands
 Cover UV box with fresh plastic wrap
 Always use oil overlay

To eliminate contamination/carryover:

 UV irradiation: Mix all components, except template DNA; irradiate in clear 0.5 ml polypropylene tubes
in direct contact with glass transilluminator (254 and 300 nm UV bulbs) for 5 minutes.
 UNG Digestion: Incorporate dUTP nucleotides into reaction and do subsequent uracyl DNA
glycosylase digestion.

4. Primer annealing temperature is too low

Primer annealing temperature is typically +50 to +60°C (may be higher or lower based on primer sequence
and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases 
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
5. Mispriming caused by secondary structure of template, snapback, or excessive homology at 3´ ends
of primers
Suggestions:

 Increase initial denaturation temperature to +95 to +97°C.

 Denature DNA minus enzyme and buffer for 4 - 6 minutes.
 Increase cycling denaturation time 15 - 30 seconds.
 Try Hot Start technique.
 Add T4 Gene 32 protein 3 - 5 µl / ml.
  When designing primers, make sure there is no more than 2 bases of homology at the 3´ end. Use a
primer design program if available.
 Consider addition of cosolvent to reaction buffer:
o 3 - 15% DMSO
o 1 - 10% formamide
o 5 - 15% polyethylene glycol
o 10 - 15% glycerol

6. DNase activity (indicated by smears visible on gel below expected band size)
Suggestions:

 Make fresh stock solutions.

 Check the integrity of template DNA.

7. Oil contamination of gel sample

Primer annealing temperature is typically +50 to +60°C (may be higher or lower based on primer sequence
and buffer components).
Suggestion:Spin the reaction tube and carefully extract the oil layer from the surface.
8. Incorrect template to enzyme ratio
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human
DNA (105 - 106 copies) per 100 µl reaction. The amount of enzyme should be optimized for each template.
Suggestions:

 Titrate the amount of template in the reaction.

 Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range
(0.5 to 5.0 units).

Low yield
Here are some troubleshooting hints that we have gathered regarding low yield after PCR reactions.
1. Primer annealing temperature is too high
Primer annealing temperature is typically +50 to +60°C (may be higher or lower based on primer sequence
and buffer components).
Suggestion: Determine Tm/annealing temperature based on the following equations:
If primers are 20-35 bases
Tp = 22 + 1.46 (Ln)
Ln = 2(# G or C) + (# A or T)
Tp = Effective annealing temperature ± 2 - 5
If primers are 14 - 70 bases 
Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12
[J+] = concentration of monovalent cations
l = length of oligo
2. Template not clean or degraded
For example, protease contamination can degrade the polymerase.
Suggestions:

 Try Hot Start technique.

 Purify the DNA template to the highest degree possible, including proteinase K digest.

3. An enzyme inhibitor is present in the reaction

Known inhibitors of PCR include:

 50 mM ammonium chloride
 EDTA (metal chelator)
 >0.8 µM hematin
 PBS (phosphate will bind free magnesium)
 >0.02 % sarcosyl
 0.5 M urea
 >5% DMF
 >10% formamide
 Heparin
 >20% PEG deoxycholate
 >0.01% SDS (can be reversed with equal molar ratio of NP40 and Tween 20)
 >10% DMSO
 >20% glycerol
 >0.4% N-octylglucoside
 Residual phenol
 >0.06% sodium deoxycholate
 Pyrophosphate

Suggestion: Reduce or remove the concentration of any inhibitor in the reaction mixture.

4. Not enough template in the reaction
The necessary amount of template varies from reaction to reaction. As a guideline, use 100 - 750 ng human
DNA (105 - 106 copies) per 100 µl reaction. The amount of enzyme should be optimized for each template.
Suggestions:

 Titrate the amount of template in the reaction.

 Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range
(0.5 to 5.0 units).

5. Extension temperature too high

Optimal extension temperature and time depends on fragment size:
  +72°C for 20 seconds for fragments <500 bp.
 +72°C for 40 seconds for fragments 1.2 bp.

Suggestions: Longer times, not higher temperatures should be used when longer templates or suspected
secondary structure is present.
6. Enzyme activity is low
For Roche polymerases, 100% activity is guaranteed through the control date.
Suggestions:

 Check the control date. If necessary, obtain fresh enzyme.

 Try Hot Start technique to help retain activity through thermal cycling.

7. Cycle number is too high

Most templates require 25 - 30 cycles.
Suggestion: Cycle number should be based on starting concentration of template DNA.
If the number of target molecules in Then we recommend the following
your sample is... number of cycles...
3 x 10 5
25 - 30
1.5 x 10 4
30 - 35
1.0 x 10 3
35 - 40
50 40 - 45
8. Nucleotides hydrolyzed
Always store nucleotide stock solutions at a concentration of at least 10 mM, 100 mM is best. Significant
hydrolysis occurs after storing at 1 mM for 2 months.
Dissolve NTPs or dNTPs in water at an expected concentration of 10 mM. Using 0.05 M Tris-base and pH
paper, adjust the pH to 7.0. Dilute an aliquot of the neutralized NTP or dNTP appropriately and read the optical
density at the wavelengths given in the following table. Calculate the actual concentration using the values for
the extinction coefficients. Freeze away in small aliquots at -20°C.
Base Wavelength Extinction coefficients for bases e (M  cm ) -1 -1

A 259 1.54 x 10 4

T 260 7.4 x 10 3

G 253 1.37 x 10 4

C 271 9.1 x 10 3

U 262 1.0 x 10 4

The lithium and sodium salts have equivalent stability and work equally well in PCR, sequencing, and labeling
applications. Lithium salts are more soluble in ethanol than sodium salts. Thus, removal of lithium salts by
ethanol precipitation is more efficient than removal of sodium salts. Using lithium salt nucleotide preparations
reduces salt-induced artifacts and increases the legibility of sequencing gels.
9. Nucleotide concentration is too high or unbalanced
The standard concentration is 20 - 200 µM of each nucleotide.
Suggestions:

 Check the concentration of stock solutions of all nucleotides.

 Double-check the final concentrations of all nucleotides.

10. Primer concentration is too low

Suggestion: Adjust the primer concentration (0.1 - 1.0 µM of each primer is optimal).
11. Machine-based error
Suggestions:

 Calibrate the heating block and confirm actual block temperature.

 Run a diagnostic program for the particular machine - contact the machine manufacturer for specifics.

12. Plateau effect

Possible causes of the plateau effect/solutions:

 Utilization of dNTP's - dNTP concentration should be 20 - 200 µM each.

 End product pyrophosphate inhibition - reduce cycle number to 20 - 35 to reduce pyrophosphate
formation.
 Incomplete denaturation of product at high concentration - use stepwise cycling, increasing time of
denaturation in later cycles.
 Substrate excess in substrate/enzyme ratio - use stepwise cycling, increasing extension time in later
cycles or enzyme concentration.

13. Evaporation
Evaporation can lead to higher concentrations of components, which may inhibit enzyme activity. The change
in volume also leads to changes in the thermal profile inside the reaction tubes.
Suggestion: Use 100 µl mineral oil overlay/reaction.

On this page:
 Low or no amplification
 Nonspecific amplification or smears
 Sequence errors within PCR products

 Sequence errors at termini of PCR products   

 False positive amplification
 Resources
Low or no amplification

Possible causes Recommendations

DNA templates

Poor integrity  Minimize shearing and nicking of DNA during isolation. Evaluate template
DNA integrity by gel electrophoresis, if necessary.
 Store DNA in molecular-grade water or TE buffer (pH 8.0) to prevent
degradation by nucleases.

Low purity  Follow manufacturer recommendations stringently when using purification kits
to isolate template DNA. Consult the user manual and troubleshooting guides
to mitigate poor DNA quality.
 Ensure that no residual PCR inhibitors such as phenol, EDTA, and proteinase
K are present if following chemical or enzymatic DNA purification protocols.
 Re-purify, or precipitate and wash DNA with 70% ethanol, to remove residual
salts or ions (e.g., K , Na , etc.) that may inhibit DNA polymerases.
+ +

 Choose DNA polymerases with high processivity, which display high tolerance

to common PCR inhibitors carried over from soil, blood, plant tissues, etc.

Insufficient  Examine the quantity of input DNA and increase the amount if necessary.

quantity  Choose DNA polymerases with high sensitivity for amplification.
 If appropriate, increase the number of PCR cycles.

Complex targets  Choose DNA polymerases with high processivity, which display high affinity
(e.g., GC-rich or for DNA templates and are more suitable to amplify difficult targets.
secondary
structures)  Use a PCR additive or co-solvent to help denature GC-rich DNA and
sequences with secondary structures.
 Increase denaturation time and/or temperature to efficiently separate double-
stranded DNA templates.

Long targets  Check amplification length capability of the selected DNA polymerases. Use
DNA polymerases specially designed for long PCR.
  Choose DNA polymerases with high processivity, which can amplify long
targets in a shorter time.
 Reduce the annealing and extension temperatures to help primer binding and
enzyme thermostability.
 Prolong the extension time according to amplicon lengths.

Primers

Problematic  Review primer design. Use online primer design tools when appropriate.

design  Ensure that the primers are specific to the target of interest.
 Verify that the primers are complementary to the correct strands of the target
DNA.

Old primers  Aliquot primers after resuspension and store properly.

 Reconstitute fresh primer aliquots, or obtain new primers if necessary.

Insufficient  Optimize primer concentrations (usually in the range of 0.1–1 μM).

quantity  For long PCR and PCR with degenerate primers, start with a minimum
concentration of 0.5 μM.

Other reaction components

Inappropriate  Use hot-start DNA polymerases to prevent degradation of primers by the

DNA polymerase 3’→5’ exonuclease activity of proofreading DNA polymerases. Hot-start DNA
polymerases also increase yields of the desired PCR products by eliminating
nonspecific amplification.
 Alternatively, set up PCR on ice, or add DNA polymerase last to the reaction
mixture.

Insufficient  Choose DNA polymerases with high sensitivity for amplification.

quantity of DNA  Review recommendations on the amount of DNA polymerase to use in PCR,
polymerase
and optimize as necessary.
 Increase the amount of DNA polymerase if the reaction mixture contains a high
concentration of an additive (e.g., DMSO, formamide) or inhibitors from the
sample sources.
Insufficient  Optimize Mg  concentration for maximum PCR yields. The presence of
2+

Mg concentration
2+
EDTA, other metal chelators, or atypically high concentrations of dNTPs may
require a higher Mg concentration.
2+

 Check the DNA polymerase’s preference for magnesium salt solutions. For
example, Pfu DNA polymerase works better with MgSO  than with MgCl .
4 2

Excess PCR  Review the recommended concentrations of PCR additives or co-solvents. Use

additives or co- the lowest possible concentration when appropriate.
solvents
 Adjust the annealing temperatures, as high concentrations of PCR additives or
co-solvents weaken primer binding to the target.
 Increase the amount of DNA polymerase, or use DNA polymerases with high
processivity.
 Consider using an additive or co-solvent specifically formulated for a given
DNA polymerase (e.g., GC Enhancer supplied with Invitrogen™ Platinum™
DNA polymerases).

dUTP or modified  Ensure that the selected DNA polymerases are able to incorporate the modified
nucleotides in nucleotides.
reaction mix
 Optimize the ratio of the modified nucleotide to dNTP to increase PCR
efficiency.

Nonhomogeneous  Mix the reagent stocks and prepared reactions thoroughly to eliminate density
reagents gradients that may have formed during storage and setup.

Thermal cycling conditions

Suboptimal  Optimize the DNA denaturation time and temperature. Short denaturing times

denaturation and low temperatures may not separate double-stranded DNA templates well.
On the other hand, long denaturation times and high temperatures may reduce
enzyme activity.

Suboptimal  Optimize the annealing temperature stepwise in 1–2°C increments, using

annealing a gradient cycler when available. The optimal annealing temperature is usually
3–5°C below the lowest primer T .m

 Adjust the annealing temperature when a PCR additive or co-solvent is used.

  Use the annealing temperature recommended for a specific DNA polymerase in
its optimal buffer. Annealing temperature rules for primer sets can vary
between different DNA polymerases.

Suboptimal  Select an extension time suitable for the amplicon length.

extension  Reduce the extension temperature (e.g., to 68°C) to keep the enzyme active
during amplification of long targets (e.g., >10 kb).
 Use DNA polymerases with high processivity for robust amplification even
with short extension times.

Suboptimal  Adjust the number of cycles (generally to 25–35 cycles) to produce an

number of PCR adequate yield of PCR products. Extend the number of cycles to 40 if DNA
cycles
input is fewer than 10 copies.

Nonspecific amplification or smears

Possible causes Recommendations

DNA templates

Excess DNA input  Review the optimal amounts of DNA input. Lower the quantity to reduce the
generation of nonspecific PCR products.

Poor integrity  Degraded DNA may appear as smears or lead to high background in gel
electrophoresis. Minimize shearing and nicking of DNA during isolation.
 Evaluate the integrity of the template DNA prior to PCR by gel
electrophoresis, if necessary. Store DNA in molecular-grade water or TE
buffer (pH 8.0) to prevent degradation by nucleases.

Complex  Choose DNA polymerases with high processivity, which display high affinity
sequences (e.g., for DNA templates and are more suitable to amplify difficult targets.
GC-rich or
secondary  Use a PCR additive or co-solvent to help denature GC-rich DNA and
structures) sequences with secondary structures. Increase denaturation time and/or
 temperature to efficiently separate double-stranded DNA templates.

Long targets  Check amplification length capability of the selected DNA polymerases. Use
DNA polymerases specially designed for long PCR.
 Choose DNA polymerases with high processivity, which can amplify long
targets in a shorter time.
 Reduce the annealing and extension temperatures to help primer binding and
enzyme thermostability. Prolong the extension time according to amplicon
lengths.

Primers

Problematic  Review primer design. Use online primer design tools when appropriate.

design Verify that the primers are specific to the target, with minimal homology to
other regions in the template.
 Ensure that the primers do not contain complementary sequences or
consecutive G or C nucleotides at the 3′ ends, to prevent primer-dimer
formation.
 Avoid direct repeats in the primers to prevent misalignment in binding to the
target. Consider longer primers to enhance specificity.
 Consider nested PCR to improve specificity.

High quantity  Optimize primer concentrations (usually in the range of 0.1–1 μM). High

primer concentrations promote primer-dimer formation.

Other reaction components

Excess DNA  Review recommendations on the amount of DNA polymerase to use in PCR,

polymerase and decrease as necessary.

Inappropriate  Use hot-start DNA polymerases that have no activity at room temperature but
DNA polymerase are functional only after a high-temperature activation step, to
enhance specificity. With non–hot-start DNA polymerases, set up PCR on ice
to keep enzyme activity low.
Excess  Review Mg  concentrations and lower as appropriate to prevent nonspecific
2+

Mg concentration
2+
PCR products. Optimize Mg  concentrations for each primer set and target
2+

DNA.

Thermal cycling conditions

Insufficient  Increase denaturation time and/or temperature to efficiently separate DNA

denaturation when working with GC-rich templates and sequences with secondary
structures.

Incorrect  Use the annealing temperature recommended for a specific DNA polymerase in

annealing its optimal buffer. Annealing temperature rules for primer sets can vary
temperature
between different DNA polymerases.

Low annealing  Increase the annealing temperature to improve specificity. The optimal
temperature annealing temperature is usually no less than 3–5°C below the lowest primer
T.
m

 Optimize the annealing temperature stepwise in 1–2°C increments, using

a gradient cycler when available.
 Consider touchdown PCR to enhance specificity.

Long annealing  Shorten the annealing time to minimize primer binding to nonspecific
time sequences.

High extension  Reduce the extension temperature 3–4°C to help the DNA polymerase’s
temperature thermostability, especially for long PCR.

Insufficient  Prolong the extension time when amplifying long DNA targets.
extension time  Include a final extension step with sufficient time (5–15 minutes) to extend the
whole target.

High number of  Reduce the number of cycles, without drastically lowering the yield of the
cycles desired PCR products, to prevent accumulation of nonspecific amplicons.
Sequence errors within PCR products

Possible causes Recommendations

Low fidelity of  Use DNA polymerases with exceptionally high fidelity to generate PCR
DNA polymerase fragments for downstream applications such as cloning, sequencing, and site-
directed mutagenesis.

Excess  Review Mg  concentrations and reduce as necessary. Excessive concentrations

2+

Mg concentration
2+
favor misincorporation of nucleotides by DNA polymerases.

Unbalanced dNTP  Ensure equimolar concentrations of dATP, dCTP, dGTP, and dTTP in the
concentrations reaction. Unbalanced nucleotide concentrations increase the PCR error rate.

High number of  Reduce the number of cycles without drastically lowering the yield of the
cycles desired PCR products. High numbers of cycles increase the incorporation of
mismatched nucleotides.
 Increase the amount of input DNA when appropriate to avoid running an
excessive number of cycles.

UV-damaged  Use a long-wavelength UV (360 nm) light box to visualize fragments in gels,
DNA and limit the illumination time as much as possible.
 If using a short-wavelength (254–312 nm) light box, limit the UV illumination
to a few seconds and keep the gel on a glass or plastic plate.
 Alternatively, use dyes with longer-wavelength (less damaging) excitation to
visualize the DNA.

Sequencing error  Sequence both DNA strands to verify the reliability of sequencing results. Use
duplicate samples when appropriate.

Sequence errors at termini of PCR products

Possible causes Recommendations

Problematic  Avoid direct repeats within primer sequences, as multiple repeats may appear
primer design from sequence misalignment at the ends of the PCR products.

Low primer  Order PCR primers with purification to remove non–full-length DNA oligos,
quality which are truncated at their 5′ ends.

Contaminating  Use molecular-grade, nuclease-free reagents in PCR setup. Set up reactions on

nucleases ice to keep the activity of possible contaminating exonucleases low.

UV-damaged  Use a long-wavelength UV (360 nm) light box to visualize fragments in gels,
DNA and limit the illumination time as much as possible.
 If using a short-wavelength (254–312 nm) light box, limit the UV illumination
to a few seconds and keep the gel on a glass or plastic plate.
 Alternatively, use dyes with longer-wavelength (less damaging) excitation to
visualize the DNA.

Sequencing error  Sequence both DNA strands to verify the reliability of sequencing results. Use
duplicate samples when appropriate.

False positive amplification

Possible causes Recommendations

Problematic  Avoid primers containing complementary and self-complementary sequences,

primer design which favor primer-dimer formation and self-oligomerization and their
subsequent amplification.

Crossover  Avoid contamination of DNA in the work environment by following general

contamination recommendations for PCR setup.
Carryover  Use pipette tips with aerosol barriers. Dedicate a separate work area and
contamination decontaminate the area after each use.
 Follow PCR carryover control techniques such as dUTP incorporation with
UDG treatment.