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...

Full description

Saved in:
Bibliographic Details
Published inNucleic acids research Vol. 31; no. 21; pp. 6139 - 6147
Main Authors Kermekchiev, Milko B., Tzekov, Anatoly, Barnes, Wayne M.
Format Journal Article
LanguageEnglish
Published England Oxford University Press 01.11.2003
Oxford Publishing Limited (England)
Subjects
Binding Sites
Catalytic Domain
Cold Temperature
DNA Replication
Enzyme Stability
Gene Library
Hot Temperature
Mutagenesis - genetics
Mutation - genetics
Polymerase Chain Reaction - methods
Sequence Analysis, DNA
Taq Polymerase - chemistry
Taq Polymerase - genetics
Taq Polymerase - metabolism
Thermus - enzymology
Thermus - genetics
Thermus aquaticus
Online AccessGet full text

Cover

More Information
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.
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