Selection of specific gene probes by combined use of low-stringency PCR amplification and Southern-blot hybridization.
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.
Magdolen
U
U
Institut für Genetik und Mikrobiologie der LMU, München, Germany.
Magdolen
V
V
Schmitt
M
M
Bandlow
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D015342:DNA Probes / Q000235:genetics*; D015723:Gene Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015003:Yeasts / Q000235:genetics*
D015342:DNA Probes