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PMID: 9153325 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.

Nucleic acids research ·Vol. 25 ·No. 11 ·1997-06-01 ·Pages 2227-8

Urban A, Neukirchen S, Jaeger KE

Abstract

A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.

MeSH Terms
Animals DNA, Bacterial/chemistry Deoxyribonucleases, Type II Site-Specific/metabolism Green Fluorescent Proteins Lipase/genetics Luminescent Proteins/genetics Mutagenesis, Site-Directed Polymerase Chain Reaction/methods Pseudomonas aeruginosa/genetics Scyphozoa
Chemicals
DNA, Bacterial Luminescent Proteins Green Fluorescent Proteins Lipase CTTAAG-specific type II deoxyribonucleases Deoxyribonucleases, Type II Site-Specific GGTACC-specific type II deoxyribonucleases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Urban A
Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universität, Universitätsstrasse 150, D-44780 Bochum, Germany.
Neukirchen S
Jaeger K E
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/25.11.2227
Published
1997-06-01
Pages
2227-8
Language
English
Region
England
NLM ID
0411011
PMCID
PMC146699
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/25.11.2227 PMC Full Text View journal details
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