An Improved Method to Obtain a Large Number of Mutants in a Defined Region of DNA

This chapter discusses the improved method to obtain a large number of mutants in a defined region of deoxyribo nucleic acid (DNA). Conditions for mutagenesis of nucleic acids with sodium bisulfite have been developed, based on the well-characterized reactions of this reagent with the bases of nucleosides, nucleotides, and nucleic acids. The reaction with uracil and thymine is fully reversible, while the adduct of cytosine can undergo hydrolytic deamination to produce 5,6-dihydrouracil-6-sulfonate. An approach in which large numbers of altered molecules are produced with random mutations in single or multiple occurrences is required for certain targets. Such a method can be designed by adaptation of low frequency mutagenesis with sodium bisulfite in combination with appropriate cloning and screening. In addition to the criteria, on which other uses of random mutagenesis are based, these targets should be sequences with many putatively important sites. Mutants containing varied but specific base alterations throughout a gene would provide a sufficient choice of replacements to be compared and studied.