A Method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction.

Abstract

A simple in vitro mutagenesis method for the generation of random point mutations in a given DNA segment is described. The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Thermus aquaticus (Taq) DNA polymerase. The pool of amplified DNA fragments can then be inserted into appropriate cloning vectors to generate random mutant libraries. DNA sequence analysis of various individual clones shows that the frequency of point mutations is about 2%. The distribution of mutations within the targeted DNA region appears to be random. Both transition and transversion base substitutions can be generated. The frequency of deletion or insertion mutations is about 5% of the level of substituion mutations. The yield of mutants with single- or multiple-point mutations is >90% when the target region to be mutagenized is >300bp. This method of mutagenesis should be useful to generate random mutants for the study of structure and function of both proteins and DNA control elements.