Laboratory methods. Efficient saturation mutagenesis of a pentapeptide coding sequence using mixed oligonucleotides

Stephen A Goff, S. R. Short-Russell, J. F. Dice

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Site-directed mutagenesis using oligonucleotides that are degenerate at a specific codon was employed to construct a set of mutations in a pentapeptide sequence targeting cystosolic proteins to lysosomes during serum withdrawal. Low-temperature annealing of the mixed oligonucleotides to single-stranded phage DNA and a genetic selection for the DNA strand carrying the mutations were utilized. The use of mixed oligonucleotides by this technique provides an economical means of generating a large set of substitution mutations. A single codon can be changed to codons for most other amino acids in one step. This approach eliminates the need for restriction enzyme cleavage sites flanking the target for mutagenesis and, therefore, is useful for targeting mutations to any DNA fragment cloned into an appropriate single-stranded bacteriophage.

Original languageEnglish (US)
Pages (from-to)381-388
Number of pages8
JournalDNA
Volume6
Issue number4
Publication statusPublished - 1987
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Molecular Biology

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