Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers
| Autor: | Yvonne M. McKeown, Hans-Joachim Fritz, Marc Zabeau, Patrick Stanssens, Wilfried Kramer, Chris Opsomer |
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| Jazyk: | angličtina |
| Rok vydání: | 1989 |
| Předmět: |
Genetics
Genetic Markers Mutation Expression vector Oligonucleotide Genetic Vectors Nucleic Acid Heteroduplexes Mutagenesis (molecular biology technique) Biology medicine.disease_cause beta-Galactosidase chemistry.chemical_compound chemistry Gene Expression Regulation Lac Operon Genes Bacterial medicine Escherichia coli Site-directed mutagenesis Oligonucleotide Probes Gene Selectable marker DNA |
| Popis: | An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model. |
| Databáze: | OpenAIRE |
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