21] Isolation of the yeast calmodulin gene using synthetic oligonucleotide probes

Abstract

Publisher Summary This chapter discusses the strategy used to clone the gene encoding calmodulin from the yeast Saccharomyces cerevisiae . The approach employed was to design oligonucleotide probes based on protein sequence information and to screen a yeast genomic DNA library with the oligonucleotides. Techniques for using oligonucleotides to identify and isolate genes have been published, and quite a number of genes have been cloned successfully using established methods. The first step for using oligonucleotide probes to identify and isolate a gene is to determine which probe(s) displays the greatest specificity and, thereby, establish appropriate conditions for screening the library. This survey is most conveniently accomplished by hybridizing each probe to separate Southern blots of restriction endonuclease-digested genomic DNA at a series of different stringencies, as described in this chapter. The procedures described are used with minor modifications to isolate genes from any organism with a genome size in the same range as yeast (or smaller). The principles utilized are extended, however, to isolate specific genes from higher eukaryotic cells.