Polymerase chain reaction optimization: Difference between revisions

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{{Short description|Methods to reduce failure and increase the reliability of PCR}}

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

== 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]], [[virus]]es, 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.<ref name="balin">{{cite journal |authorvauthors=Balin BJ, Gérard HC, Arking EJ, ''et al.''etal |title=Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain |journal=Med. Microbiol. Immunol. |volume=187 |issue=1 |pages=23–42 |year=1998 |pmid=9749980 |doi= 10.1007/s004300050071|notess2cid=25307947 |quote="Extreme care was taken in all assays to avoid cross-contamination of both nucleic acid samples to be analyzed and reaction mixtures; such measures included preparation of nucleic acids in a laboratory separate from those in which PCR or reverse transcription (RT)-PCR assays were set up and use of eight different biologic hoods, each in a different laboratory, for setting up reactions." <!--NOTE: This "quote" was a "note". Someone with access to the full text of the article should verify that it actually exists in the text. It is not in the abstract.--> }}</ref> 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 procedure]]s involve using [[pipette]]s with [[filter tips]] and wearing fresh [[medical gloves|laboratory glove]]s, and in some cases a [[laminar flow cabinet]] with UV lamp as a work station (to destroy any [[extraneomultimer]] formation). PCR is routinely assessed against a [[negative control]] reaction that is set up identically to the experimental PCR, but without template DNA, and performed alongside the experimental PCR.

[[File:PCR_gel_electrophoresis.jpg|alt=A Graph of PCR Gel electrophoresis|right|thumb|PCR Gel electrophoresis]]

== Hairpins ==

[[Secondary structure]]s in the DNA can result in folding or knotting of DNA template or primers, leading to decreased product yield or failure of the reaction. [[Stem-loop|Hairpin]]s, 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 [[Dimethyl sulphoxide|DMSO]] or [[glycerol]] to the PCR to minimize secondary structures in the DNA template ,<ref>{{CitationCite neededweb|dateurl =February 2007https://www.neb.com/products/pcr-polymerases-and-amplification-technologies/taq-dna-polymerases/~/media/26059D85ADCD473DB16C5500AD69EE60.ashx|title = FAQs for Polymerases and Amplification|publisher = New England Biolabs}},</ref> are used in the optimization of PCRs that have a history of failure due to suspected DNA hairpins. http://www.promega.com/pnotes/65/6921_27/6921_27_core.pdf

== Polymerase errors ==

[[Taq polymerase]] lacks a 3'[[Directionality (molecular biology)|3′ to 5'5′]] [[exonuclease|exonuclease activity]]. Thus, Taq has no error-[[Proofreading (biology)|proof-reading activity]], which consists of excision of any newly misincorporated nucleotide base from the nascent (i.e., extending) DNA strand that does not match with its opposite base in the complementary DNA strand. The lack in 3'3′ to 5'5′ proofreading of the Taq enzyme results in a high error rate (mutations per nucleotide per cycle) of approximately 1 in 10,000{{formatnum:10000}} bases, which affects the fidelity of the PCR, especially if errors occur early in the PCR with low amounts of starting material, causing accumulation of a large proportion of amplified DNA with incorrect sequence in the final product.<ref> {{cite journal |authorvauthors= Eckert KA, Kunkel TA |title=DNA polymerase fidelity and the polymerase chain reaction |journal=PCRGenome Methods Appl.Research |volume=1 |issue=1 |pages=17–24 |yeardate=August 1991 |month=August |pmid=1842916 |doi= 10.1101/gr.1.1.17 |url= http://www.genome.org/cgi/pmidlookup?view=long&pmid=1842916 |doi-access= free }}</ref>

Several "high-fidelity" [[thermostable DNA polymerasespolymerase]]s, having engineered 3'3′ to 5'5′ exonuclease activity, have become available that permit more accurate amplification for use in PCRs for sequencing or cloning of products. Examples of polymerases with 3'3′ to 5'5′ 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''.;<ref>{{Citationcite journal needed|datetitle =February 2007High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus |journal= Gene |volume= 108 |issue= 1 |pages= 1–6 |year= 1991 |last1= Lundberg |first1= Kelly S.|last2= Shoemaker |first2= Dan D.|last3= Adams |first3= Michael W.W.|last4= Short |first4= Jay M.|last5= Sorge |first5 = Joseph A.|last6= Mathur |first6= Eric J. |doi= 10.1016/0378-1119(91)90480-y |pmid= 1761218}}</ref> Q5 polymerase, with 280x higher fidelity amplification compared with ''Taq''.<ref>New England Biolabes. "Q5® High-Fidelity DNA Polymerase." [https://international.neb.com/products/m0491-q5-high-fidelity-dna-polymerase#Product%20Information Available].</ref>

==Magnesium concentration==

Magnesium is required as a co-factor for thermostable DNA polymerase. Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction.<ref name="Markoulatos2002">{{cite journal |authorvauthors=Markoulatos P, Siafakas N, Moncany M |title=Multiplex polymerase chain reaction: a practical approach |journal=J. Clin. Lab. Anal. |volume=16 |issue=1 |pages=47–51 |year=2002 |pmid=11835531|doi=10.1002/jcla.2058|pmc=6808141 }}</ref> Some of the components of the reaction mixture such as template concentration, dNTPs and the presence of [[chelating agents]] ([[EDTA]]) or [[proteins]] can reduce the amount of free magnesium present thus reducing the activity of the enzyme.<ref name="Promega">{{cite journal|title=Nucleic acid amplification protocols and guidelines|url=http://www.promega.com/paguide/chap1.htm|access-date=2009-01-28|archive-url=https://web.archive.org/web/20090202111629/http://www.promega.com/paguide/chap1.htm|archive-date=2009-02-02|url-status=dead}}</ref> Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations and so results in decreased specificity of the reaction. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.<ref name="Markoulatos2002"/><ref name="Promega"/> Inadequate thawing of MgCl₂ may result in the formation of concentration gradients within the [[magnesium chloride]] solution supplied with the DNA polymerase and also contributes to many failed experiments Doksh .<ref name="Promega"/>

== 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 [[fusion protein|fused]] to a processivity-enhancing DNA-binding protein, enhancing adherence of the polymerase to the DNA.<ref>{{cite journal |authorvauthors=Pavlov AR, Belova GI, Kozyavkin SA, Slesarev AI |year= 2002|title=Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases|journal=Proc. Natl. Acad. Sci. U.S.A.|volume=99|pages=3510–13515|pmid=12368475|doi=10.1073/pnas.202127199 |issue=21 |pmc=129704|bibcode= 2002PNAS...9913510P|doi-access= free}}</ref><ref>{{cite journal |author=Demidov VV|year=2002|title= A happy marriage: advancing DNA polymerases with DNA topoisomerase supplements|journal=Trends Biotechnol.|volume=20|pages=491|doi=10.1016/S0167-7799(02)02101-7 |issue=12}}</ref>

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 |authorvauthors=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 |chapterurlchapter-url=http://www.horizonpress.com/hsp/abs/absdna.html|authorvauthors=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|doi-access=free}}</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|chapterurlchapter-url=http://bioscience.jbpub.com/catalog/0763733830/table_of_contents.htm |authorvauthors=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.{{factcitation 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=httphttps://www.ncbi.nlm.nih.gov/sutils/e-pcr/|publisher=NCBI - National Center for Biotechnology Information|accessdateaccess-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.

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==Primer dimers==

[[Annealing (biology)|Annealing]] of the 3' end of one [[primer (molecular biology)|primer]] to itself or the second primer may cause primer extension, resulting in the formation of so-called primer dimers, visible as low-molecular-weight bands on [[Agarose gel electrophoresis|PCR gels]].<ref name="kramer2006">{{cite journal |authorvauthors=Kramer MF, Coen DM |title=Enzymatic amplification of DNA by PCR: standard procedures and optimization |journal=Curr Protoc Cytom |volume=Appendix 3 |issue= |pages=Appendix A.3K.1–A.3K.15 |yeardate=August 2006 |month=August |pmid=18770830 |doi=10.1002/0471142956.cya03ks37 |urls2cid=4658404 }}</ref> Primer dimer formation often competes with formation of the DNA fragment of interest, and may be avoided using primers that are designed such that they lack [[complementarity (molecular biology)|complementarity]]—especially at the 3' ends—to itself or the other primer used in the reaction. If primer design is constrained by other factors and if primer-dimers do occur, methods to limit their formation may include optimisation of the MgCl₂ concentration or increasing the annealing temperature in the PCR.<ref name="kramer2006"/>

==Deoxynucleotides==

Deoxynucleotides (dNTPs) may bind Mg²⁺ ions and thus affect the concentration of free magnesium ions in the reaction. In addition, excessive amounts of dNTPs can increase the error rate of DNA polymerase and even inhibit the reaction.<ref name="Markoulatos2002"/><ref name="Promega"/> An imbalance in the proportion of the four dNTPs can result in misincorporation into the newly formed DNA strand and contribute to a decrease in the fidelity of DNA polymerase.<ref>{{cite journal |authorvauthors=Kunz BA, Kohalmi SE |title=Modulation of mutagenesis by deoxyribonucleotide levels |journal=Annu. Rev. Genet. |volume=25 |issue= |pages=339–59 |year=1991 |pmid=1812810 |doi=10.1146/annurev.ge.25.120191.002011 |url=}}</ref>

== References ==

{{reflist}}

<references/>

[[Category:Biochemistry methods]]

[[Category:DNA]]

[[Category:Molecular biology]]

[[Category:Polymerase chain reaction]]