RAPID AMPLIFICATION OF UNCHARACTERIZED TRANSPOSON-TAGGED DNA-SEQUENCES FROM GENOMIC DNA

Although the entire DNA sequence of the yeast genome has been determin ed, the functions of nearly a third of the identified genes are unknow n. Recently, we described a collection of mutants, each with a transpo son-tagged disruption in an essential gene in Saccharomyces cerevisiae . Identification of these essential genes and characterization of thei r mutant phenotypes should help assign functions to these thousands of novel genes, and since each mutation in our collection is physically marked by the uniform, unique DNA sequence of the transposable element , it should be possible to use the polymerase chain reaction (PCR) to amplify the DNA adjacent to the transposon. However, existing PCR meth ods include steps that make their use on a large scale cumbersome. In this report, we describe a semi-random, two-step PCR protocol, ST-PCR. This method is simpler and more specific than current methods, requir ing only genomic DNA and two pairs of PCR primers, and involving two s uccessive PCR reactions. Using this method, we have rapidly and easily identified the essential genes identified by several of our mutants. (C) 1997 by John Wiley & Sons, Ltd.