Polymerase chain reaction optimization

The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization have been developed by molecular biologists to improve PCR performance and minimize failure.

Contamination and PCR

Since PCR is very sensitive, i.e., requiring only a few DNA molecules for amplification across several orders of magnitude, adequate measures to avoid contamination from any DNA present in the lab environment (bacteria, viruses, lab staff's skin etc.) should be taken. Thus DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should all be performed in separate areas. Because products from previous PCR amplifications are a common source of contamination, one area should be dedicated to reaction assembly before the PCR and another area to post-PCR processing, such as electrophoresis or purification of PCR products. For the preparation of reaction mixtures, a laminar flow cabinet with UV lamp is recommended, and pipettes with filter tips should be used. Fresh gloves should be used for each PCR step as well as displacement pipettes with aerosol filters. The reagents for PCR should be prepared separately and used solely for this purpose. Aliquots should be stored separately from other DNA samples. A control reaction, omitting template DNA (also called negative control), should always be performed alongside experimental PCRs, to check for possible contamination of reagents with extraneous DNA or for primer multimer formation.

Hairpins

Secondary structures in the DNA, caused by base-pairing between nucleotides on the same strand of the molecule, can cause folding or even knotting of the DNA template or the primers, leading to decreased yield or total failure of the reaction. Hairpins, direct folding of the DNA caused by a run of complementary bases or an inversion, are the most common problems of this sort.

Typically, this calls for choosing different primers; secondary structures in the template DNA are not as serious as those in the primers, as the DNA polymerase will "flatten out" most secondary structures unless they are particularly robust.

However, if use of hairpin-forming primers is necessary, as may be the case in PCR splicing and cloning, the problem can be ameliorated somewhat by use of DMSO or glycerol ; these chemicals can be added to the PCR mastermix to interrupt secondary structures.

Polymerase errors

Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to do error proofreading, i.e., check the last base it has inserted and excise it if the base does not match with the base in the complementary strand. This lack in 3' to 5' proofreading results in a high error rate of approximately 1 in 10,000 bases, which, if an error occurs early in the PCR, can cause accumulation of a large proportion of amplified DNA with incorrect sequence in the final product. Several "high-fidelity" DNA polymerases, having engineered 3' to 5' exonuclease activity have become available that permit more accurate amplification for use in amplification for sequencing or cloning. Examples of polymerases with 3' to 5' exonuclease activity include: KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis KOD1; Vent, which is extracted from Thermococcus litoralis; Pfu DNA polymerase, which is extracted from Pyrococcus furiosus; and Pwo, which is extracted from Pyrococcus woesii.

Size and other limitations

PCR works readily with DNA of up two to three thousand base pairs in length. However, above this size, product yields often decrease, as with increasing length stochastic effects such as premature termination by the polymerase begin to affect the efficiency of the PCR. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These are polymerases fused to a processivity-enhancing DNA-binding protein, making them literally "stick" to the DNA longer.

Other valuable properties of the prototype chimeric polymerases TopoTaq and PfuC2 include enhanced thermostability, specificity and resistance to contaminants and inhibitors. They were engineered using unique Helix-hairpin-Helix (HhH) DNA binding domains of Topoisomerase V from hyperthermophile Methanopyrus kandleri. Chimeric polymerases overcome many limitations of native enzymes and are used in direct PCR amplification from cell cultures and even food samples, thus by-passing laborious DNA isolation steps altogether. A robust strand displacement activity of the hybrid TopoTaq polymerase helps solving PCR problems with #Hairpins and G-loaded double helices, because helices with a high G-C context possess a higher melting temperature .

Non-specific priming

Non-specific binding of primers frequently occurs and can be due to repeat sequences in the DNA template, non-specific binding between primer and template, and incomplete primer binding, leaving the 5' end of the primer unattached to the template. Non-specific binding is also often increased when degenerate primers are used in the PCR. Manipulation of annealing temperature and magnesium ion (which stabilise DNA and RNA interactions) concentrations can increase specificity. Non-specific priming during reaction preparation at lower temperatures can be prevented by using "hot-start" polymerase enzymes whose active site is blocked by an antibody or chemical that only dislodges once the reaction is heated to 95˚C during the denaturation step of the first cycle.

A new way to maintain thermophilic enzymes absolutely inactive at low temperature was identified during structural studies of hyperthermophilic DNA-binding enzymes. A specially engineered TopoTaq polymerase activates instantly at high temperature and overcomes limitations of conventional "hot-start" enzymes that require antibody denaturation at >90˚C for activation. In addition, its activity is blocked upon completion of PCR at low temperature.

Other methods to increase specificity include Nested PCR and Touchdown PCR.