Single-stranded gaps as localized targets for in vitro mutagenesis.

David R Shortle, D. Botstein

Research output: Contribution to journalArticle

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 beta-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 C1aI 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.

Original languageEnglish (US)
Pages (from-to)147-155
Number of pages9
JournalBasic Life Sciences
Volume20
StatePublished - 1982
Externally publishedYes

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Mutagenesis
Cytosine
DNA
Micrococcus luteus
DNA Ligases
Circular DNA
DNA Polymerase I
Deamination
Mutation
Adenine
beta-Lactamases
Genes
In Vitro Techniques
sodium bisulfite
triphosphoric acid

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Single-stranded gaps as localized targets for in vitro mutagenesis. / Shortle, David R; Botstein, D.

In: Basic Life Sciences, Vol. 20, 1982, p. 147-155.

Research output: Contribution to journalArticle

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