A simplified lysis method allowing the use of biotinylated probes in colony hybridization.
A method is described for the lysis of bacterial cells grown on nitrocellulose filters which allows the use of nonradioactive (biotin labeled) probes in colony hybridization. Used in conjunction with a colorimetric assay involving streptavidin and alkaline phosphatase this lysis method allows the detection of clones containing a target nucleic acid sequence. Sites of positive hybridization produce dark blue-purple signals, while nonreacting clones give very light blue signals. The occurrence of false-positive and background signals is minimal. With pBR322 as the target sequence it was possible to detect approximately 20 clones in the presence of 10(5) plasmid-free cells. It was also possible to detect low-frequency plasmid-free cells in a population of clones containing the target sequence.
Haas
M J
MJ
Eastern Regional Research Center, US Department of Agriculture, Philadelphia, Pennsylvania 19118.
Fleming
D J
DJ
D001433:Bacteriolysis; D001710:Biotin / Q000494:pharmacology*; D002999:Clone Cells / Q000032:analysis*; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D006824:Hybridization, Genetic; D010957:Plasmids
D004269:DNA, Bacterial; D001710:Biotin