Polymerase chain reaction optimization: Difference between revisions

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== Polymerase errors ==

[[Taq polymerase]] lacks a [[Directionality (molecular biology)|3′ to 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′ to 5′ proofreading of the Taq enzyme results in a high error rate (mutations per nucleotide per cycle) of approximately 1 in {{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 |vauthors= 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 |date=August 1991 |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" 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]]''; Pwo, which is extracted from ''Pyrococcus woesii'';<ref>{{cite journal |title = High-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>