Randomization of genes by PCR mutagenesis.

TLDR

A modified PCR was developed to introduce random point mutations into cloned genes. The method reduces Taq polymerase fidelity while maintaining amplification, enabling cloning of random mutant libraries or direct transcription when a T7 promoter primer is used. The technique produced a mutation rate of 0.66 % ± 0.13 % per position per PCR, with no bias toward particular base substitutions, and mutations followed a Poisson distribution and were randomly distributed across the sequence.

Abstract

A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes. The modifications were made to decrease the fidelity of Taq polymerase during DNA synthesis without significantly decreasing the level of amplification achieved in the PCR. The resulting PCR products can be cloned to produce random mutant libraries or transcribed directly if a T7 promoter is incorporated within the appropriate PCR primer. We used this method to mutagenize the gene that encodes the Tetrahymena ribozyme with a mutation rate of 0.66% +/- 0.13% (95% C.I.) per position per PCR, as determined by sequence analysis. There are no strong preferneces with respect to the type of base substituion. The number of mutations per DNA sequence follows a Poisson distribution and the mutations are randomly distributed throughout the amplified sequence.