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Enzymological description of multitemplate PCR-Shrinking amplification bias by optimizing the polymerase-template ratio

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dc.title Enzymological description of multitemplate PCR-Shrinking amplification bias by optimizing the polymerase-template ratio en
dc.contributor.author Ingr, Marek
dc.contributor.author Dostál, Jiří
dc.contributor.author Majerová, Taťána
dc.relation.ispartof Journal of Theoretical Biology
dc.identifier.issn 0022-5193 Scopus Sources, Sherpa/RoMEO, JCR
dc.date.issued 2015
utb.relation.volume 382
dc.citation.spage 178
dc.citation.epage 186
dc.type article
dc.language.iso en
dc.publisher Academic Press
dc.identifier.doi 10.1016/j.jtbi.2015.06.048
dc.relation.uri https://www.sciencedirect.com/science/article/pii/S0022519315003276
dc.subject Amplicons pooling en
dc.subject Amplification efficiency en
dc.subject Library preparation en
dc.subject Processivity en
dc.subject Species diversity en
dc.description.abstract Multitemplate polymerase chain reaction (PCR) is used for preparative and analytical applications in diagnostics and research. Classical PCR and qPCR are two basic setups with many possible experimental modifications. Classical PCR is a method of choice to obtain enough material for subsequent sophisticated applications such as construction of libraries for next-generation sequencing or high-throughput screening. Sequencing and Single Nucleotide Primer Extension (SNuPE) employ one-strand synthesis and represent a distinct variant of analytical DNA synthesis. In all these applications, maintaining the initial ratio of templates and avoiding underestimation of minority templates is desired. Here, we demonstrate that different templates can amplify independently at low template concentrations (typical in qPCR setups, in which the polymerase concentration is usually several orders of magnitude higher than the template concentration). However, rare templates can be diluted in an effort to keep DNA amplification in the exponential phase, or template concentration can be biased by differences in amplification efficiency. Moreover, amplification of templates present in low concentrations is more vulnerable to stochastic events that lead to proportional changes in the product ratio, as well as by incomplete amplification leading to chimera formation. These undesired effects can be compensated for by using highly processive polymerases with high and equal affinity to different primer-template complexes. Novel enhanced polymerases are desired. With increasing concentration of a primer-template of interest, the system becomes more deterministic. Nevertheless, marked deviation from independent exponential amplification occurs when the total template concentration starts to approach the polymerase concentration. The primer-template complexes compete for enzyme molecules, and the amount of products grows arithmetically-the system starts to obey Michaelis-Menten kinetics. Synthesis of rare products in a multitemplate mixture can run more easily under the detection limit in such conditions, although it would be unequivocally detectable in a single template assay. When fishing out rare template variants, the best processive polymerases should be used to decrease both amplification and detection limits. The possibility of stochastic events, should be taken into account to correctly interpret the obtained data. © 2015 Elsevier Ltd. en
utb.faculty Faculty of Technology
dc.identifier.uri http://hdl.handle.net/10563/1005258
utb.identifier.obdid 43873440
utb.identifier.scopus 2-s2.0-84937848180
utb.identifier.wok 000361085200017
utb.identifier.pubmed 26164060
utb.identifier.coden JTBIA
utb.source j-scopus
dc.date.accessioned 2015-08-28T12:04:45Z
dc.date.available 2015-08-28T12:04:45Z
dc.description.sponsorship Ministry of Education of the Czech Republic [LO 1302]; Grant Agency of the Czech Republic [P208-12-G016]; Gilead Sciences; IOCB Research Centre
utb.contributor.internalauthor Ingr, Marek
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