Efficient site directed in vitro mutagenesis using ampicillin selection.
A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.
Lewis
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MK
Promega Corporation, Madison, WI 53711.
Thompson
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DV
D000668:Ampicillin Resistance / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D013698:Templates, Genetic
D009838:Oligodeoxyribonucleotides