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PMID: 2154748 Published · ppublish English Journal Article

High-efficiency oligonucleotide-directed plasmid mutagenesis.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 4 ·1990-02-00 ·Pages 1451-5

Olsen DB, Eckstein F

Abstract

A number of single- and double-base substitutions have been introduced into either the polylinker region or the lacZ gene in the plasmid vector pUC19. The efficiencies of these changes upon transfection of TG-1 bacterial cells were generally 70-80%. A strategy has been devised by which the wild-type DNA can be selectively destroyed. It is primarily based on the resistance of phosphorothioate internucleotide linkages to some restriction enzymes. A mismatch oligonucleotide is introduced into a gapped region and the gap is filled using three deoxynucleoside 5'-triphosphates and one deoxynucleoside 5'-[alpha-thio]triphosphate. Reaction with a restriction enzyme that is unable to hydrolyze phosphorothioates ensures that the DNA containing the mismatch oligonucleotide is only nicked. Concomitantly, the DNA that does not contain the desired mutation is linearized. Subsequent reactions with an exonuclease and DNA polymerase I yield mutant homoduplex DNA for transfection.

MeSH Terms
Base Sequence DNA Restriction Enzymes/metabolism Molecular Sequence Data Mutation Nucleic Acid Heteroduplexes/genetics Oligonucleotide Probes/chemical synthesis Plasmids Restriction Mapping
Chemicals
Nucleic Acid Heteroduplexes Oligonucleotide Probes DNA Restriction Enzymes
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Olsen D B
Max-Planck Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Eckstein F
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.87.4.1451
Published
1990-02-00
Pages
1451-5
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC53493
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.87.4.1451 PMC Full Text View journal details
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