Versatile PCR-mediated insertion or deletion mutagenesis

Abstract

based procedure that can be used to generate precisely located mutants with any length of insertion or deletion. The strategy for insertion mutagenesis employs an established overlap extension PCR protocol, but involves three templates including an insert fragment of any length. The protocol for insertion requires four chimeric primers that are derived partly from the sequence to be inserted and partly from the template, in addition to two outermost flanking primers. Three PCR products are first prepared separately, including one insert fragment and two adjacent fragments using appropriate primer pairs. The insert fragment thus generated contains terminal complementarities at both ends to two adjacent fragments. In the second step, an overlap extension PCR is carried out, using the outermost primer