Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene.

TLDR

The method enables isolation of any gene represented by an available RNA sequence. Colonies are grown on nitrocellulose filters, lysed, and their DNA is fixed and hybridized to a radioactive RNA probe; autoradiography identifies positive colonies, which are then selected from the reference plate. The technique successfully isolated ColE1 hybrid plasmids carrying Drosophila melanogaster 18S and 28S rRNA genes.

Abstract

A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.