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PMID: 6092841 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Oligonucleotide directed mutagenesis: selection of mutants by hemimethylation of GATC-sequences.

Molecular & general genetics : MGG ·Vol. 195 ·No. 1-2 ·1984-00-00 ·Pages 126-33

Marmenout A, Remaut E, van Boom J, Fiers W

Abstract

We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an AT----CG transversion in the mut L region of phage lambda. Under optimal conditions, about 50-60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.

MeSH Terms
Bacteriophage lambda/genetics Base Sequence DNA Restriction Enzymes DNA, Viral/genetics Escherichia coli/genetics Genotype Methylation Mutation Nucleic Acid Hybridization Oligodeoxyribonucleotides/pharmacology Oligonucleotides/pharmacology Plasmids Species Specificity
Chemicals
DNA, Viral Oligodeoxyribonucleotides Oligonucleotides DNA Restriction Enzymes
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Marmenout A
Remaut E
van Boom J
Fiers W
References (35)
35 references, click to expand
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Article Info
Journal
Molecular & general genetics : MGG
Abbr.
Mol Gen Genet
ISSN
0026-8925
DOI
10.1007/BF00332734
Published
1984-00-00
Pages
126-33
Language
English
Region
Germany
NLM ID
0125036
Subset
IM
External Links
PubMed source DOI: 10.1007/BF00332734
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