Colony or Plaque Hybridization for Screening of Libraries, genomic Library, gene Sequence, Colohny Hybridization, Technique Bacteria, Nitrocellulose Filters

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Home >> Biotechnology and Genomics >> Chimeric DNA Molecular Probes and Gene Libraries >> Colony or Plaque Hybridization for Screening of Libraries

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Colony (or plaque) hybridization for screening of libraries

Once a genomic library or cDNA library is available we may like to use it for isolation of a gene sequence. This can be achieved by colony hybridization technique illustrated. In this technique, bacteria carrying chimeric vectors are grown into colonies, which are lysed on nitrocellulose filters.

Their DNA is denatured in situand fixed on the filter which is hybridized with a radioactively labelled probe carrying a sequence related to the gene to be isolated (usually a cloned cDNA for screening of a genomic library).

Colony hybridization technique for isolation and isolation of a DNA fragment

Colonies carrying this sequence will be identified by dark spots after autoradiography, so that the original chimeric vector carrying the desired gene sequence can be recovered from one or more colonies in the original master plate and used for further experiments. This technique is described as colony hybridization. It is possible that a probe may identify more than one clones or that a gene is fragmented in the library.

In such a case one needs to reconstruct the desired sequence using several overlapping sequences available in the library. This is a very routine exercise whenever we like to isolate specific DNA sequences from the genome of a species, or from cDNA derived from mRNA of a specific tissue of a species.

Sometimes the library may be available not in the form of bacteria transformed with chimeric DNA molecules but in the form of chimeric phage particles carrying the cloned segments. In such a situation, a bacterial lawn is infected with a mixture of chimeric phage particles (i.e. the library) and a large number of plaques develop overnight. These plaques can be treated just like the colonies in colony hybridization to identify and isolate the chimeric phage particles carrying the gene of interest. This technique is then described as plaque hybridization.

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