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.

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Essential Laboratory Skills Guide

Contamination and PCR

The PCR method is extremely sensitive, requiring only a few DNA molecules in a single reaction for amplification across several orders of magnitude. Therefore, adequate measures to avoid contamination from any DNA present in the lab environment (bacteria, viruses, or human sources) are required. Because products from previous PCR amplifications are a common source of contamination, many molecular biology labs have implemented procedures that involve dividing the lab into separate areas.^[1] One lab area is dedicated to preparation and handling of pre-PCR reagents and the setup of the PCR reaction, and another area to post-PCR processing, such as gel electrophoresis or PCR product purification. For the setup of PCR reactions, many standard operating procedures involve using pipettes with filter tips and wearing fresh laboratory gloves, and in some cases a laminar flow cabinet with UV lamp as a work station (to destroy any extraneous DNA before PCR setup). Possible contamination with extraneous DNA or primer-multimer formation is routinely assessed with a (negative) control PCR reaction. This control reaction is set up in the same way as the experimental PCRs, but without template DNA added, and is performed alongside the experimental PCRs.

Hairpins

Secondary structures in the DNA can result in folding or knotting of DNA template or primers, leading to decreased product yield or failure of the reaction. Hairpins, which consist of internal folds caused by base-pairing between nucleotides in inverted repeats within single-stranded DNA, are common secondary structures and may result in failed PCRs.

Typically, primer design that includes a check for potential secondary structures in the primers, or addition of DMSO or glycerol to the PCR to minimize secondary structures in the DNA template^{[citation needed]}, are used in the optimization of PCRs that have a history of failure due to suspected DNA hairpins.

Polymerase errors

Taq polymerase lacks a 3' to 5' exonuclease activity. Thus, Taq has no error-proofreading activity, which consists of excision of any newly polymerized nucleotide base from the nascent (=extending) DNA strand that does not match with its opposite base in the complementary DNA strand. The lack in 3' to 5' proofreading of the Taq enzyme results in a high error rate (approximately 1 in 10,000 bases), which affects the fidelity of the PCR, especially if errors occur early in the PCR, causing 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 PCRs for sequencing or cloning of products. 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.^{[citation needed]}

Size and other limitations

PCR works readily with a DNA template of up to 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, enhancing adherence of the polymerase to the DNA ^[2][3].

Other valuable properties of the chimeric polymerases TopoTaq and PfuC2 include enhanced thermostability, specificity and resistance to contaminants and inhibitors^[4][5]. They were engineered using the unique helix-hairpin-helix (HhH) DNA binding domains of topoisomerase V^[6] 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. 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 ^[7].

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^[8]. 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.

References

1. ^ Balin BJ, Gérard HC, Arking EJ, et al (1998). "Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain". Med. Microbiol. Immunol. 187 (1): 23–42. PMID 9749980.
2. ^ Pavlov AR, Belova GI, Kozyavkin SA, Slesarev AI (2002). "Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases". Proc Natl Acad Sci. 99: 3510-13515. PMID 12368475.
3. ^ Demidov VV (2002). "A happy marriage: advancing DNA polymerases with DNA topoisomerase supplements". Trends Biotechnol. 20: 491. doi:10.1016/S0167-7799(02)02101-7.
4. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends Biotechnol. 22: 253-260. PMID 15109812.
5. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Thermostable Chimeric DNA Polymerases with High Resistance to Inhibitors", DNA Amplification: Current Technologies and Applications. Horizon Bioscience, pp. 3-20. ISBN 0-9545232-9-6. 
6. ^ Forterre P (2006). "DNA topoisomerase V: a new fold of mysterious origin". Trends Biotechnol. 24: 245-247. PMID 16650908.
7. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0. 
8. ^ Taneja B, Patel A, Slesarev A, Mondragon A (2006). "Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases". EMBO J. 25: 398-408. PMID 16395333.