25] Colony hybridization

Abstract

Publisher Summary This chapter discusses colony hybridization, which allows bacterial clones to be rapidly screened by ribonucleic acid–deoxyribonucleic acid (RNA–DNA) or DNA–DNA hybridization for those containing specific DNA sequences. Bacterial colonies are grown on nitrocellulose filters lying on agar petri plates. Reference sets of colonies are made by replica plating and stored at 2°–4°. The cells on the filter are lysed, and their DNA is denatured in situ without dislodging the remnants of the cells from their initial site. Radiolabeled RNA or DNA probe is then hybridized with the filters. After washing away unhybridized probe, autoradiography reveals the DNA that prints on the filter and the colonies in the reference set that contain the DNA sequence in question. It is now also possible to screen phage plaques in a similar manner, with the added advantages of lambda phage cloning. It is possible to combine the advantages of lambda and plasmid cloning by the partial lysis of bacteria lysogenic for a thermoinducible phage. This allows a large number of colonies to be screened and obviates the need for replica plating a reference set. The colony hybridization procedure has also been adapted for the screening of yeast transformants and animal cell SV40-induced plaques.