Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)233-240
Number of pages8
JournalYeast
Volume13
Issue number3
DOIs
StatePublished - Mar 15 1997

Fingerprint

Polymerase chain reaction
DNA sequences
transposons
Amplification
DNA
Genes
polymerase chain reaction
genomics
nucleotide sequences
Polymerase Chain Reaction
Essential Genes
Yeast
mutants
DNA Transposable Elements
methodology
Saccharomyces cerevisiae
genes
Yeasts
Genome
yeasts

Keywords

  • PCR
  • Saccharomyces cerevisiae
  • transposon

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Bioengineering
  • Microbiology

Cite this

Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. / Chun, Kristin; Edenberg, Howard; Kelley, Mark; Goebl, Mark.

In: Yeast, Vol. 13, No. 3, 15.03.1997, p. 233-240.

Research output: Contribution to journalArticle

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