Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.

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Abstract
Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. Two in vitro reaction schemes are described that produce a high frequency of errors during repair (misrepair) of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the 1st of these misrepair schemes, nucleotide misincorporations are stimulated by omission of 1 of the 4 deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The 2nd misrepair scheme involves the misincorporation of an excision-resistant .alpha.-thiophosphate nucleotide, followed by gap filling in the presence of all 4 conventional deoxynucleoside triphosphates. When applied to short gaps constructed at 1 of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6-42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of 1-4 base pairs.