Single-Stranded Gaps as Localized Targets for in Vitro Mutagenesis
- 1 January 1982
- book chapter
- Published by Springer Nature
- Vol. 20, 147-156
- https://doi.org/10.1007/978-1-4613-3476-7_8
Abstract
Short single-stranded gaps in circular DNA molecules can be generated enzymatically, often at predetermined sites. These can serve as targets for in vitro mutagenesis procedures that result in alterations in nucleotide sequence within or very near the gap. Deamination of unpaired cytosine residues with sodium bisulfite has been used to induce mutations in the BglI restriction site of SV40 DNA and within defined regions of the β-lactamase gene on pBR322. A new method of induction of mutations at gaps, called “gap misrepair,” has been developed; it was used to cause changes at the HindIII and ClaI restriction sites on pBR322 DNA. Gap misrepair reactions using DNA polymerase I of Micrococcus luteus in the presence of T4 DNA ligase and three of the four deoxynucleoside triphosphates yielded all three possible substitutions for adenine and cytosine residues in the DNA.Keywords
This publication has 13 references indexed in Scilit:
- Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.Proceedings of the National Academy of Sciences, 1980
- Regulatory mutants of simian virus 40: Constructed mutants with base substitutions at the origin of DNA replicationJournal of Molecular Biology, 1979
- Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli.Proceedings of the National Academy of Sciences, 1979
- Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments.Proceedings of the National Academy of Sciences, 1979
- On the Fidelity of DNA ReplicationPublished by Cold Spring Harbor Laboratory ,1979
- Site-directed mutagenesis in DNA: Generation of point mutations in cloned β globin complementary DNA at the positions corresponding to amino acids 121 to 123Journal of Molecular Biology, 1978
- Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.Proceedings of the National Academy of Sciences, 1978
- Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis.Proceedings of the National Academy of Sciences, 1977
- Bisulfite Modification of Nucleic Acids and their ConstituentsProgress in Nucleic Acid Research and Molecular Biology, 1976
- [23] The use of the T4 DNA polymerase in identification of 3′ terminal nucleotide sequences of duplex DNAPublished by Elsevier ,1974