PMID: 8610108Apr 2, 1996Paper

Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops

Proceedings of the National Academy of Sciences of the United States of America
G TomblineV Sgaramella

Abstract

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.

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Citations

Apr 28, 2004·Trends in Biotechnology·Andrey R PavlovAlexei I Slesarev
Dec 1, 1996·Nature Biotechnology·J A FergusonD R Walt
Nov 20, 2002·European Journal of Immunogenetics : Official Journal of the British Society for Histocompatibility and Immunogenetics·M LongeriG Damiani
Aug 15, 2012·Molekuliarnaia biologiia·I V BotezatuA V Likhtenshtein
May 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·V LyamichevJ E Dahlberg
Sep 20, 2015·Analytical Biochemistry·Irina V BotezatuAnatoly V Lichtenstein
Nov 16, 2004·Biophysical Journal·James H WernerPeter M Goodwin
Sep 6, 2011·Analytical Biochemistry·Irina V BotezatuAnatoly V Lichtenstein
Jul 20, 2002·BMC Biotechnology·Shumo LiuAlbert Libchaber
Aug 5, 2008·Clinical Chemistry·Luming ZhouCarl T Wittwer

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