When coupled to natural transformation in Acinetobacter sp. strain ADP1, PCR mutagenesis is made less random by mismatch repair.
Random PCR mutagenesis is a powerful tool for structure-function analysis of targeted proteins, especially when coupled with DNA integration through natural transformation followed by selection for loss of function. The technique has been applied successfully to structure-function analysis of transcriptional regulators, enzymes, and transporters in Acinetobacter sp. strain ADP1. However, the mismatch repair system prevents the full spectrum of nucleotide substitutions that may be selected at the level of protein function from being recovered. This barrier may be overcome by introducing PCR-mutagenized genes into strains in which the corresponding genes have been deleted.
Buchan
Alison
A
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA.
Ornston
L Nicholas
LN
D000150:Acinetobacter / Q000235:genetics*; D020137:Base Pair Mismatch; D004260:DNA Repair; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D014169:Transformation, Bacterial
D051719:MutS DNA Mismatch-Binding Protein