A general method for saturation mutagenesis of cloned DNA fragments

Academic Article

Abstract

  • A new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA. The resulting duplex DNA fragments containing random single-base substitutions are cloned, amplified as a population, and isolated from wild-type DNA by preparative denaturing gradient gel electrophoresis. The physical separation of mutant DNA fragments makes it possible to isolate and characterize large numbers of site-directed single-base substitutions in the absence of a phenotypic selection. This procedure should be generally applicable to the fine-structure genetic analysis of regulatory and protein-coding sequences.
  • Digital Object Identifier (doi)

    Author List

  • Myers RM; Lerman LS; Maniatis T
  • Start Page

  • 242
  • End Page

  • 247
  • Volume

  • 229
  • Issue

  • 4710