Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme.
Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.
Tsurushita
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N
Department of Genetics, Stanford University School of Medicine, CA 94305.
Maki
H
H
Korn
L J
LJ
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D004258:DNA Polymerase III / Q000378:metabolism*; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009154:Mutation
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase