Molecular diversity and catalytic activity of Thermus DNA polymerases.
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.
Gibbs
Moreland D
MD
Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, Sydney, NSW, 2109, Australia. moreland.gibbs@mq.edu.au
Reeves
Rosalind A
RA
Mandelman
David
D
Mi
Qingli
Q
Lee
Jun
J
Bergquist
Peter L
PL
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D014644:Genetic Variation; D010802:Phylogeny; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013824:Thermus / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D004256:DNA Polymerase I