Selection of specific gene probes by combined use of low-stringency PCR amplification and Southern-blot hybridization

Curr Genet. 1995 Mar;27(4):390-2. doi: 10.1007/BF00352110.

Abstract

We have developed a protocol to isolate a gene from which only limited (amino-acid) sequence information is available. It involves two PCR amplifications using one constant primer and a set of nested primers and subsequent crosswise Southern hybridization. The amplified DNA giving a signal in both lanes is further processed for use in gene bank screening by applying standard procedures. In this way the structural gene for a thiol protease, BLH1, the homologue of the bleomycin A (a cancerostatic drug) resistance gene of rabbit (and man), was isolated from yeast genomic DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Southern*
  • Cloning, Molecular / methods
  • DNA Probes / genetics*
  • Gene Library
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Yeasts / genetics*

Substances

  • DNA Probes