A simplified lysis method allowing the use of biotinylated probes in colony hybridization

• Published in: Analytical biochemistry Vol. 168; no. 2; p. 239
• Main Authors: Haas, M J, Fleming, D J
• Format: Journal Article
• Language: English
• Published: United States 01-02-1988
• Subjects: Bacteriolysis; Biotin - pharmacology; Clone Cells - analysis; DNA, Bacterial; Escherichia coli - genetics; Hybridization, Genetic; Plasmids
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(88)90313-2

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.