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PMID: 8041621 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase.

Nucleic acids research ·Vol. 22 ·No. 13 ·1994-07-11 ·Pages 2587-91

Picard V, Ersdal-Badju E, Lu A, Bock SC

Abstract

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.

MeSH Terms
DNA-Directed DNA Polymerase/metabolism Mutagenesis Polymerase Chain Reaction/methods
Chemicals
Pfu DNA polymerase DNA-Directed DNA Polymerase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Picard V
Temple University School of Medicine, Department of Microbiology and Immunology, Philadelphia, PA 19140.
Ersdal-Badju E
Lu A
Bock S C
References (10)
10 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/22.13.2587
Published
1994-07-11
Pages
2587-91
Language
English
Region
England
NLM ID
0411011
PMCID
PMC308213
Subset
IM
Grants
NHLBI NIH HHS · HL45486 · United States
NHLBI NIH HHS · R01-HL30712 · United States
External Links
PubMed source DOI: 10.1093/nar/22.13.2587 PMC Full Text View journal details
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