Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA.
Although the entire DNA sequence of the yeast genome has been determined, the functions of nearly a third of the identified genes are unknown. Recently, we described a collection of mutants, each with a transposon-tagged disruption in an essential gene in Saccharomyces cerevisiae. Identification of these essential genes and characterization of their 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 methods 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, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. Using this method, we have rapidly and easily identified the essential genes identified by several of our mutants.
Chun
K T
KT
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202, USA.
Edenberg
H J
HJ
Kelley
M R
MR
Goebl
M G
MG
D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics*; D005800:Genes, Fungal / Q000502:physiology*; D016681:Genome, Fungal; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004251:DNA Transposable Elements; D004271:DNA, Fungal