Generation of multiple site-specific mutations in a single polymerase chain reaction product

Abstract

Site-directed mutagenesis has been very valuable in studying the structure-function relationship of proteins (1). PCR-mediated mutagenesis has been the method of choice in recent years to change the desired amino acid from one to another. Several procedures have been described to achieve the desired mutation using PCR technology (2-13). Although in most of the cases the desired change is only with respect to one amino acid, sometimes one needs to generate several changes in the amino acid sequence to understand the biological function of a given protein. Such a necessity is apparent when one is dealing with a zinc finger class of proteins wherein multiple residues of cysteine and histidine are involved in the coordination of zinc. We have been working over the past several years on the structure-function relationship of a spermatidal specific protein TP2 which appears transiently during mammalian spermiogenesis.