Oligonucleotide-directed mutagenesis: a sequence-based screening

Anal Biochem. 1988 Nov 1;174(2):618-22. doi: 10.1016/0003-2697(88)90064-4.

Abstract

A procedure to screen for mutant clones obtained by oligonucleotide-directed mutagenesis has been developed. It is based on the preparation of phage containing supernatants from a number (100 or more) of randomly chosen mutagenized M13 plaques. Aliquots from these supernatants are mixed to obtain pools, each containing 10 phages. Heterogeneous single-stranded DNA is prepared from these pools and used as template in a "single letter" sequence according to the dideoxy chain terminator method. Thus, the pool(s) containing the mutated sequence and the mutated sequence itself is identified by sequencing the single-stranded DNAs of the 10 phages present in the selected pool.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Clone Cells / physiology
  • Codon
  • Mutation* / drug effects*
  • Oligonucleotides / pharmacology*
  • RNA, Transfer, Pro / genetics

Substances

  • Codon
  • Oligonucleotides
  • RNA, Transfer, Pro