Efficient site directed in vitro mutagenesis using ampicillin selection
| Autor: | David V. Thompson, Martin K. Lewis |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
DNA polymerase
Genetic Vectors Molecular Sequence Data Plasmid Amp resistance Genetics Escherichia coli Cloning Molecular Phosphorylation Site-directed mutagenesis chemistry.chemical_classification DNA ligase biology Base Sequence Mutagenesis Templates Genetic Molecular biology chemistry Oligodeoxyribonucleotides Genes Bacterial Mutation biology.protein Primer (molecular biology) In vitro recombination Ampicillin Resistance Plasmids |
| Zdroj: | Nucleic acids research. 18(12) |
| ISSN: | 0305-1048 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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