Molecular diversity and catalytic activity of Thermus DNA polymerases

Show simple item record Gibbs, MD en Reeves, RA en Mandelman, D en Mi, QL en Lee, J en Bergquist, Peter en 2011-07-24T21:45:53Z en 2009-09 en
dc.identifier.citation Extremophiles 13(5):817-826 Sep 2009 en
dc.identifier.issn 1431-0651 en
dc.identifier.uri en
dc.description.abstract Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s. We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22 representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5′–3′ exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction. en
dc.language EN en
dc.publisher SPRINGER TOKYO en
dc.relation.ispartofseries EXTREMOPHILES en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. Details obtained from en
dc.rights.uri en
dc.subject Thermus en
dc.subject Enzymes en
dc.subject DNA polymerase en
dc.subject Thermophile en
dc.subject ESCHERICHIA-COLI en
dc.subject CALDOPHILUS GK24 en
dc.subject ENCODING GENE en
dc.subject CLONING en
dc.subject AMPLIFICATION en
dc.subject PURIFICATION en
dc.subject REPLICATION en
dc.title Molecular diversity and catalytic activity of Thermus DNA polymerases en
dc.type Journal Article en
dc.identifier.doi 10.1007/s00792-009-0269-8 en
pubs.issue 5 en
pubs.begin-page 817 en
pubs.volume 13 en
dc.rights.holder Copyright: 2009 Springer en
dc.identifier.pmid 19597696 en
pubs.end-page 826 en
dc.rights.accessrights en
pubs.subtype Article en
pubs.elements-id 186638 en
pubs.record-created-at-source-date 2011-11-02 en
pubs.dimensions-id 19597696 en

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