[46] Detection and isolation of novel protein-tyrosine kinase genes employing reduced stringency hybridization

Publisher Summary This chapter describes the process of detecting and isolating novel protein-tyrosine kinase (PTK) genes employing reduced stringency hybridization. This process requires a high sensitivity and specificity of genomic Southern blot analysis. By using a slightly modified procedure, a human single-copy genomic DNA fragments is detected with a completely matching probe at high stringency after 2–4 hr of film exposure at –70 ° in the presence of intensifier screens. Ten micrograms genomic DNA is restricted and to monitor restriction, bacteriophage λ DNA is incubated with a small aliquot of the main reaction at identical DNA/enzyme unit ratio. After complete restriction, DNA samples are purified with an equal volume of buffered phenol/CIA (chloroform/isoamyl alcohol, 24:1) and precipitated from 1 M ammonium acetate with 2.5 vol cold ethanol on dry ice for 20 min. Cross-hybridization of related tyrosine kinase gene fragments is accomplished by reduced stringency hybridization facilitating hybrid formation and stability between partially mismatched sequences. To determine experimentally the optimal stringency for cross-hybridization of a novel related tyrosine kinase gene, it proves useful to subject replica nitrocellulose filters containing genomic DNA to hybridization under stringencies incrementally decreased by 7°.