Troubleshooting your PCR

Consider the following:

• First, ensure that all PCR components were included in the reactions. A positive control should always be included to ensure that each component is present and functional.
• If there were no problems with the experimental setup, increase the number of PCR cycles (3–5 cycles at a time), up to 40 cycles. Increasing the cycle number can overcome issues with a low-abundance template or template inaccessibility due to impurities in or poor priming efficiency of the primers.
• If increasing the cycle number does not improve results, the PCR conditions might be too stringent for the particular primer set or template. Consider modifying the PCR conditions as follows:
  • Lower the annealing temperature in increments of 2 degrees.
  • Increase the extension time.
  • Increase the template amount. Refer to the guidelines provided with the enzyme to determine the optimal amount of template.

Consider these additional possible reasons for PCR failure:

• PCR inhibitors in the template sample 
  If PCR inhibitors are present, using diluted template may increase PCR efficiency. Alternatively, the template may need to be purified using a kit such as the NucleoSpin Gel and PCR Clean-up kit. If purifying the template is not a possibility, an enzyme that has a higher tolerance to impurities, such as Terra PCR Direct polymerase, may improve results.

• The template has >65% GC content 
  When amplifying from templates with high GC content, use an enzyme formulated for this condition. Visit our PCR selection guide to find an appropriate enzyme.

• Primers are not optimal 
  Check your primers carefully; redesign if necessary. Also, consider re-amplifying the primary PCR product using 10-fold dilutions (1:100 to 1:10,000) using nested primers.

When using PrimeSTAR HS DNA Polymerase, consider:

• Using an appropriate amount of template. If the template is human genomic DNA or a cDNA library, use no more than ~100 ng of the template in a 50-µl reaction mixture.
• Using an extension time of at least 1 min/kb.
• Increasing the concentration of the primers.

When using PrimeSTAR Max DNA Polymerase, consider:

• Adjusting the extension time if the reaction mixture contains excess template. If the amount of template exceeds 200 ng in a 50-µl reaction mixture, set the extension time between 30 sec/kb and 1 min/kb.
• Increasing the concentration of the primers.

When using SpeedSTAR HS DNA Polymerase, consider:

• Increasing the extension time. Although the standard extension time is 10 sec/kb, the extension time can be increased to ~0.5 min/kb for complex templates such as human genomic DNA.

All Takara Bio PCR polymerases

Issue:

Primers are not specific.

Solution:

Use BLAST alignment to determine if the 3' ends of the primers are complementary to sites other than the target site(s). Redesign primers if necessary or modify PCR conditions.

Issue:

PCR conditions are not sufficiently stringent.

Solutions:

• Increase the annealing temperature in increments of 2 degrees.
• Use touchdown PCR.
• Use a two-step PCR protocol.
• Reduce the number of PCR cycles.

Issue:

Too much template was used.

Solution:

Reduce the amount by 2–5 fold.

PrimeSTAR HS and PrimeSTAR Max DNA polymerases

Issue:

Annealing time is too long.

Solution:

To achieve specific amplification, it is essential to use a short annealing time (5–15 sec) when performing three-step PCR.

PrimeSTAR GXL DNA polymerases

Issue:

Primers have suboptimal T_m values.

Solution:

To amplify targets <1 kb, design primers with T_m values >55°C, and use an annealing temperature of 60°C. If the primer T_m values are <55°C, try a shorter extension time between 5 and 10 sec/kb.

Takara Ex Taq and Takara LA Taq DNA polymerases

Issue:

Nonspecific primer annealing at low temperatures.

Solution:

The hot-start versions of these enzymes may improve results for some primers.

SpeedSTAR HS DNA Polymerase

Issue:

Smearing of the PCR product bands on a gel.

Solution:

Excessively long extension times may result in smearing. The general recommendation for extension time for this enzyme is 10–20 sec/kb. If PCR yield is low, try increasing the number of cycles by 5.

First, determine the source of the smear using positive and negative (no template) controls. This can determine if the cause of the smear is contamination or overcycling, or if the smear results from poorly designed primers or suboptimal PCR conditions.

If the negative control is blank, there is no contamination. Instead, the PCR conditions will need to be optimized; consider the following when adjusting the PCR conditions:

• Reduce the amount of template.
• Increase the annealing temperature.
• Use touchdown PCR.
• Reduce the number of PCR cycles.
• Redesign the primers.
• Use nested primers.
• Re-amplify the product. (A small plug of the gel can be removed with a micropipette tip, and the DNA can be recovered by adding the plug to 200 µl of water and then incubating at 37°C. 5 µl of this solution can be used as PCR template for re-amplification.)

If the negative control is also smeared, there is contamination. You will need to determine the source of this contamination. It may be necessary to replace PCR reagents and to decontaminate pipettes and your workstation (see questions below for more information on contamination).

There are four main sources of PCR contamination:

1. The most common source of contamination is PCR product from previous amplifications (called "carryover contamination"). When large amounts of PCR product (10¹² molecules) are generated repeatedly over a period of time, the potential for contamination increases.
2. Another source of contamination is cloned DNA previously handled in the laboratory.
3. Sample-to-sample contamination can also occur. This source of contamination is most likely to be found in samples that require extensive processing prior to amplification.
4. Reactions can also be contaminated with exogenous DNA in the environment, including DNA present on laboratory equipment and in reagents used for DNA extraction and PCR.

The sensitivity of PCR requires that samples are not contaminated with any exogenous DNA or any previously amplified products from the laboratory environment. We recommend that distinct areas are used for sample preparation, PCR setup, and post-PCR analysis.

A laminar flow cabinet equipped with a UV lamp is recommended for preparing reaction mixtures. Two stations should be established that are physically separated from each other.

• Establish a "pre-PCR area" that is for PCR reaction setup only. No items from the "post-PCR area" should be introduced into this area; this includes items such as notebooks and pens.
• Establish a "post-PCR area" that is used for PCR, purifying PCR-amplified DNA, measuring DNA concentration, running agarose gels, and analyzing PCR products.

Equipment should also be restricted to these areas. The PCR machine and electrophoresis apparatus should be located in the post-PCR area. Having pipettes and pipette tips with aerosol filters dedicated for DNA sample and reaction mixture preparation only is strongly recommended. Additional recommendations include:

• Having separate sets of pipettes and pipette tips, lab coats, glove boxes, and waste baskets for the pre-PCR and post-PCR areas.
• Labeling pre- and post-PCR items, so they are not removed from their designated work area.
• Following the golden rule of PCR: NEVER bring any reagents, equipment, or pipettes used in a post-PCR area back to the pre-PCR area.
• Preparing and storing reagents for PCR separately and using them solely for their designated purpose. Reagents should be aliquoted in small portions and stored in designated areas depending on if they are used for pre-PCR or post-PCR applications. The aliquots should be stored separately from other DNA samples.

A control reaction that omits template DNA should always be performed to confirm the absence of contamination. In addition, the number of PCR cycles should be kept to a minimum, as highly sensitive assays are more prone to the effects of contamination.

1. Leave pipettes under UV light in the cell culture hood overnight. UV irradiation promotes cross-linking of thymidine residues, damaging residual DNA.
2. Spray workstations/equipment/pipettes with 10% bleach and then wipe clean.
3. Change workstations; move the pre-PCR area to another pre-cleaned location.
4. Do not use any instruments or pipettes you have used before.

To avoid errors during PCR, we recommend using a high-fidelity enzyme. In addition, be sure to avoid the following:

1. Overcycling
   Overcycling PCR reactions often:
   • Changes the pH of the reaction in a manner that destabilizes DNA.
   • Increases the amount of PCR product, thereby reducing the efficiency of the polymerase and promoting errors.
   • Decreases the amount of dNTPs, thereby increasing the likelihood of base misincorporation due to the unbalanced dNTP concentration. (If using Takara Bio's PCR enzymes, the dNTP concentration is optimized to 200 nM; increasing dNTP concentration leads to misincorporation.)
   • Causes accumulation of single-stranded and double-stranded DNA.
2. High Mg²⁺ concentration
   Mg²⁺ concentration ranges from 1–5 mM. Using a high Mg²⁺ concentration may increase yield, but it might also impact the proofreading activity of enzymes. However, the Mg²⁺ concentration should always be higher than the dNTP concentration.
3. Template DNA damage
   Limit UV exposure time when analyzing or excising PCR products from gels.

Impurities that interfere with PCR amplification are known as PCR inhibitors. PCR inhibitors are present in a large variety of sample types and may lead to decreased PCR sensitivity or even false-negative PCR results. PCR inhibitors may have both inorganic and organic origins (Schrader 2012).

Inorganic PCR inhibitors include:

• Calcium or other metal ions that compete with magnesium
• EDTA that binds to magnesium, reducing its concentration

Some organic PCR inhibitors include:

• Polysaccharides and glycolipids that mimic the structure of nucleic acids, interfering with primers binding to the template
• Melanin and collagen that form a reversible complex with DNA polymerase
• Humic acids that interact with template DNA and polymerase, preventing the enzymatic reaction, even at low concentrations
• Urea that may lead to degradation of the polymerase

Other organic compounds that can inhibit PCR include:

• Hemoglobin, lactoferrin, and IgG in blood, serum, or plasma samples
• Anticoagulants such as heparin
• Polyphenols, pectin, and xylane from plants
• Ethanol, isopropyl alcohol, phenol, or detergents such as SDS

If inhibitors are present in the template preparation, a 100-fold dilution of the starting template may sufficiently dilute the inhibitor and allow amplification. Alternatively, ethanol precipitation of the template may be needed to resolve the problem.

References

Schrader, C., et al. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. 113:1014–1026 (2012).

PCR overcycling is when cycling goes beyond the exponential phase of amplification. Overcycling occurs when the following events take place during PCR:

• Depletion of substrates (dNTPs or primers).
• The reagents (dNTPs or enzymes) are no longer stable at the denaturation temperature.
• The PCR polymerase is inhibited by the product (pyrophosphate, duplex DNA).
• Competition for reagents (dNTPs and primers) by nonspecific products.
• Lowering of the pH of the reaction.
• Incomplete denaturation/strand separation of products at high product concentrations.

The indicator of PCR overcycling is an intense background smear with indistinguishable bands when the reaction is resolved on an agarose gel. It is always recommended to perform a preliminary test to determine the minimal number of PCR cycles needed to yield a sufficient product. The PCR product remains in the linear phase of amplification if the product yield is noticeably increased every 3–5 cycles. We find that overcycled cDNA does not produce suitable template for any downstream application.

PCR polymerases can introduce different types of mutations, including single-base substitutions, deletions, and insertions. Base substitutions are typically caused by misincorporation of an incorrect dNTP during DNA synthesis.

Polymerases may generate mutations at locations where one or more nucleotides are lost or gained. The frequency of this type of mutation can be sequence dependent, and might be higher in highly repetitive sequences. The most common mutation is a loss of a single nucleotide, which could be a result of template-primer misalignment within a repetitive homopolymeric sequence. DNA rearrangements can also occur when the polymerase terminates synthesis on one DNA strand and continues synthesis after priming occurs on a complementary strand (i.e., strand-switching or jumping PCR). This type of mutation takes place when there is high homology between different regions of DNA. Excessive DNA template in the reaction may also promote this type of mutation.

The following factors can contribute to PCR-introduced mutations:

• Unbalanced dNTP concentrations
  Unequal amounts of the four dNTPs can increase base substitution by as high as eight-fold. Using equal concentrations of the four dNTPs is critical for reducing the error rate of the polymerase.
• High enzyme concentration

• Long incubation times

• Lack of 3'→5' exonuclease activity

• Magnesium concentration
  Fidelity is highest when the concentration of Mg²⁺ is equimolar to the total concentration of the dNTPs. Fidelity decreases when the concentration of free divalent cations increases.

• pH of the reaction
  Lowering the pH of the reaction by three units can increase base substitutions up to 60-fold. Low pH (<6.0) may lead to spontaneous purine loss.

• DNA damage
  DNA damage can occur at high temperatures, possibly increasing the rate of mutation. One frequent mutation is deamination of cytosine to produce uracil.

• The presence of A stretches in primer sequences

• Overcycling
  Error rate is increased when the DNA concentration is increased during the final PCR cycles. The total number of cycles should be kept to a minimum to produce the desired PCR product without errors.

The following are common PCR artifacts:

• Primer dimers
  Primer dimers are formed through self-complementarity at the 3' end of the amplification primers. Primer dimers are suspected if product is produced in a template-free reaction (negative control). To avoid primer dimers, primers shouldn't have complementarity at their 3' ends.
• Chimeric PCR products
  Chimeric PCR products can be caused by incompletely extended template. In other words, single-stranded template that was not completely replicated due to premature polymerase termination can anneal to partially homologous template. This creates chimeric PCR products. To minimize chimeras, use the fewest possible PCR amplification cycles.
• PCR bias
  PCR bias occurs when some sequences are amplified more efficiently than others due to preferential binding by PCR primers. If one sequence is amplified 10% more than another in one cycle, it will be 17.4-times more abundant after 30 cycles. To reduce PCR bias, use a high ramp rate between the denaturation and annealing steps and use low annealing temperatures. Long extension times (>180 sec) should be avoided.
• PCR drift
  PCR drift is due to stochastic fluctuation in the interactions of PCR reagents, particularly in the early cycles when a very low template concentration exists. This artifact is observed in multiplex assays, where a loss of sensitivity is caused by the interactions between different sets of primers. It is important to carefully design primers for these types of assays.
• PCR generates high-molecular-weight products that barely migrate through the agarose gel
  There is no good explanation for this artifact. Most researchers assume that this is caused by overcycling, since in the later stages of PCR, both single- and double-stranded molecules accumulate. Accumulation of such single-stranded molecules can create heteroduplexes by competing with the primers. Incomplete denaturation in later stages, when there is a high concentration of PCR products, prevents DNA strand separation, and thus a newly formed amplicon may remain bound to the previously made template. This process could repeat, trapping PCR products in a network of molecules.
  
  Another explanation for the origin of high-molecular-weight smears is the partial extension of templates during initial PCR cycles. Partial extensions could be generated by jumping artifacts—when a primer or single-stranded DNA anneals and extends from one priming site, then anneals partially to a homologous segment elsewhere (see Chimeric PCR products, above). Partially extended molecules can act as new primers, since they contain a free 3'-OH, and could generate chimeric molecules that combine the initial priming site and the "jump" site.
  
  Finally, this type of artifact can also be generated when a crude template is used for PCR. Products amplified directly from animal or plant tissues can become trapped in cell debris, which prevents them from migrating in the gel. This problem can be solved by Proteinase K digestion of the amplified PCR product:
  
  
  • Add 15 µl of loading buffer containing Proteinase K to the entire 50-µl PCR reaction.
  
  OR
  
  
  • Before loading your samples onto a gel, add 1 µl of the loading buffer containing Proteinase K to 4 µl of the PCR reaction.