Cold‐sensitive mutants of Taq DNA polymerase provide a hot start for PCR
Although the thermophilic bacterium Thermus aquaticus grows optimally at 70°C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20–37°C. This activity is a bane to some PCRs, since it catalyzes non‐specific priming. We report mutations of Klentaq (an N‐termin...
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Published in | Nucleic acids research Vol. 31; no. 21; pp. 6139 - 6147 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.11.2003
Oxford Publishing Limited (England) |
Subjects | |
Online Access | Get full text |
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Summary: | Although the thermophilic bacterium Thermus aquaticus grows optimally at 70°C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20–37°C. This activity is a bane to some PCRs, since it catalyzes non‐specific priming. We report mutations of Klentaq (an N‐terminal deletion variant) DNA polymerase that have markedly reduced activity at 37°C yet retain apparently normal activity at 68°C and resistance at 95°C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold‐sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity. |
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Bibliography: | Received as resubmission July 27, 2003; Accepted September 4, 2003 ark:/67375/HXZ-G3TZ5T9T-M local:gkg813 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA. Tel: +1 314 362 3351; Fax: +1 253 369 3024; Email: wayne@barnes1.wustl.edu Correspondence may also be addressed to Milko B. Kermekchiev. Tel: +1 314 771 5566; Fax: +1 314 771 5581; Email: milko@klentaq.com istex:CCDE0D006BD9F92B740529CA5B0AA6DB5755C7B2 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA. Tel: +1 314 362 3351; Fax: +1 253 369 3024; Email: wayne@barnes1.wustl.edu Correspondence may also be addressed to Milko B. Kermekchiev. Tel: +1 314 771 5566; Fax: +1 314 771 5581; Email: milko@klentaq.com |
ISSN: | 0305-1048 1362-4962 1362-4962 |
DOI: | 10.1093/nar/gkg813 |