PCR Troubleshooting

If standard PCR conditions do not yield the desired amplicon, PCR optimization is necessary to attain better results. The stringency of a reaction may be modulated such that the specificity is adjusted by altering variables (e.g., reagent concentrations, cycling conditions) that affect the outcome of the amplicon profile. For example, if the reaction is not stringent enough, many spurious amplicons will be generated with variable lengths. If the reaction is too stringent, no product will be produced. Troubleshooting PCR reactions may be a frustrating endeavor at times. However, careful analysis and a good understanding of the reagents used in a PCR experiment can reduce the amount of time and trials needed to obtain the desired results. Of all the considerations that impact PCR stringency, titration of Mg2+ and/or manipulating annealing temperatures likely will solve most problems. However, before changing anything, be sure that an erroneous result was not due to human error. Start by confirming all reagents were added to a given reaction and that the reagents were not contaminated. Also take note of the erroneous result, and ask the following questions: Are primer dimers visible on the gel after electrophoresis (these run as small bands <100 b near the bottom of the lane)? Are there non-specific products (bands that migrate at a different size than the desired product)? Was there a lack of any product? Is the target DNA on a plasmid or in a genomic DNA extract? Also, it is wise to analyze the G-C content of the desired amplicon.

First determine if any of the PCR reagents are catastrophic to your reaction. This can be achieved by preparing new reagents (e.g., fresh working stocks, new dilutions), and then systematically adding one new reagent at a time to reaction mixtures. This process will determine which reagent was the culprit for the failed PCR experiment. In the case of very old DNA, which often accumulates inhibitors, it has been demonstrated that addition of bovine serum albumin may help alleviate the problem.
Primer dimers can form when primers preferentially self anneal or anneal to the other primer in the reaction. If this occurs, a small product of less than 100 bp will appear on the agarose gel. Start by altering the ratio of template to primer; if the primer concentration is in extreme excess over the template concentration, then the primers will be more likely to anneal to themselves or each other over the DNA template. Adding DMSO and or using a hot start thermal cycling method may resolve the problem. In the end it may be necessary to design new primers.
Non-specific products are produced when PCR stringency is excessively low resulting in non-specific PCR bands with variable lengths. This produces a ladder effect on an agarose gel. It then is advisable to choose PCR conditions that increase stringency. A smear of various sizes may also result from primers designed to highly repetitive sequences when amplifying genomic DNA. However, the same primers may amplify a target sequence on a plasmid without encountering the same problem.
Lack of PCR products is likely due to reaction conditions that are too stringent. Primer dimers and hairpin loop structures that form with the primers or in the denatured template DNA may also prevent amplification of PCR products because these molecules may no longer base pair with the desired DNA counterpart.
If the G-C content has not been analyzed, it is time to do so. PCR of G-C rich regions (GC content >60%) pose some of the greatest challenges to PCR. However, there are many additives that have been used to help alleviate the challenges.

REFERENCES
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies