Polymerase chain reaction optimization: Difference between revisions

Revision as of 06:31, 24 April 2021

Revision as of 06:31, 24 April 2021

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Other valuable properties of the chimeric polymerases [http://www.fidelitysystems.com/TopoTaq.html TopoTaq] and PfuC2 include enhanced thermostability, specificity and resistance to contaminants and [[polymerase chain reaction inhibitors|inhibitors]].<ref>{{cite journal |vauthors=Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI |year= 2004|title=Recent developments in the optimization of thermostable DNA polymerases for efficient applications|journal= Trends Biotechnol.|volume=22|pages= 253–260|pmid= 15109812|doi=10.1016/j.tibtech.2004.02.011 |issue=5}}</ref><ref>{{cite book |chapter-url=http://www.horizonpress.com/hsp/abs/absdna.html|vauthors=Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI |year=2004|chapter=Thermostable Chimeric DNA Polymerases with High Resistance to Inhibitors|title=DNA Amplification: Current Technologies and Applications|publisher=Horizon Bioscience|pages=3–20|isbn=0-9545232-9-6}}</ref> They were engineered using the unique [[helix-hairpin-helix]] (HhH) DNA binding domains of [[topoisomerase]] V<ref>{{cite journal |author=Forterre P|year= 2006|title=DNA topoisomerase V: a new fold of mysterious origin|journal= Trends Biotechnol.|volume=24|pages= 245–247|pmid=16650908|doi=10.1016/j.tibtech.2006.04.006 |issue=6}}</ref> 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 sampling|food samples]], thus by-passing laborious DNA isolation steps. A robust strand-displacement activity of the hybrid TopoTaq polymerase helps solve PCR problems that can be caused by [[Stem-loop|hairpins]] and [[Gyromagnetic ratio|G-loaded]] double helices. Helices with a high G-C content possess a higher melting temperature, often impairing PCR, depending on the conditions.<ref name=Hybrid>{{cite book|chapter-url=http://bioscience.jbpub.com/catalog/0763733830/table_of_contents.htm |vauthors=Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI |year= 2006|chapter=Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes|title=DNA Sequencing II: Optimizing Preparation and Cleanup|editor=Kieleczawa J|publisher=Jones and Bartlett|pages=241–257|isbn=0-7637-3383-0}}</ref>

== Non-specific priming ==

Non-specific binding of primers frequently occurs and may occur for several reasons. These include repeat sequences in the DNA template, non-specific binding between primer and template, high or low G-C content in the template, or incomplete primer binding, leaving the 5' end of the primer unattached to the template. Non-specific binding of [[Primer (molecular biology)|degenerate primers]] is also common. Manipulation of [[Annealing (biology)|annealing]] temperature and [[magnesium]] ion concentration may be used to increase specificity. For example, lower concentrations of magnesium or other cations may prevent non-specific primer interactions, thus enabling successful PCR. A "hot-start" polymerase enzyme whose activity is blocked unless it is heated to high temperature (e.g., 90–98˚C) during the [[Denaturation (biochemistry)|denaturation]] step of the first cycle, is commonly used to prevent non-specific priming during reaction preparation at lower temperatures. Chemically mediated hot-start PCRs require higher temperatures and longer incubation times for polymerase activation, compared with antibody or aptamer-based hot-start PCRs.{{citation needed|date=July 2012}}

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

Computer simulations of theoretical PCR results ([[In silico PCR|Electronic PCR]]) may be performed to assist in primer design.<ref name=ePCR>{{cite web|title=Electronic PCR|url=https://www.ncbi.nlm.nih.gov/sutils/e-pcr/|publisher=NCBI - National Center for Biotechnology Information|access-date=13 March 2012}}</ref>

Touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers will avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will "swamp out" any specific sequences because of the exponential nature of polymerase amplification.

The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles (the number of individual cycles and increments of temperature decrease is chosen by the experimenter). The primer will anneal at the highest temperature which is least-permissive of nonspecific binding that it is able to tolerate. Thus, the first sequence amplified is the one between the regions of greatest primer specificity; it is most likely that this is the sequence of interest. These fragments will be further amplified during subsequent rounds at lower temperatures, and will out compete the nonspecific sequences to which the primers may bind at those lower temperatures. If the primer initially (during the higher-temperature phases) binds to the sequence of interest, subsequent rounds of polymerase chain reaction can be performed upon the product to further amplify those fragments.

==Primer dimers==