[31] Localized Mutagenesis 457 [31] Directed Mutagenesis with Sodium Bisulfite

With the development of recombinant DNA technology and the many analytic techniques based on restriction endonucleases, it has become relatively routine to isolate a segment of genomic DNA carrying a gene of interest, to define the limits of the gene within this DNA segment, and then to determine the gene's complete nucleotide sequence. In this way, a number of structural questions can be directly answered. To extend the analysis of a gene further, particularly with regard to functional and regulatory phenomena, requires the isolation and systematic study of a sizable collection of mutant alleles of the gene. In the initial stages of such a mutational analysis, deletion and insertion mutations are often useful to identify important functional elements of the gene, particularly those at the 5' and 3' ends. When base substitution mutations are required to extend the analysis, directed mutagenesis with sodium bisulfite can provide an in oitro method for efficiently inducing C to T transition mutations at sites in a DNA molecule specified in advance by the experimenter.