4-5.2.1(b).Plaque hybridization

Plaque hybridization, also known as Plaque lift, was developed by Benton and Davis in 1977 and employs a filter lift method applied to phage plaques. This procedure is successfully applied to the isolation of recombinant phage by nucleic acid hybridization and probably is the most widely applied method of library screening. The method of screening library by plaque hybridization is described below-

• The nitrocellulose filter is applied to the upper surface of agar plates, making a direct contact between plaques and filter.

•  The plaques contain phage particles, as well as a considerable amount of unpackaged recombinant DNA which bind to the filter.

•  The DNA is denatured, fixed to the filter, hybridized with radioactive probes and assayed by autoradiography.

 

Advantages

•  This method results in a ‘cleaner' background and distinct signal (less background probe hybridization) for λ plaque screening due to less DNA transfer from the bacterial host to the nitrocellulose membrane while lifting plaques rather than bacterial colonies.

•  Multiple screens can be performed from the same plate as plaques can be lifted several times.

•  Screening can be performed at very high density by screening small plaques. High-density screening has the advantage that a large number of recombinant clones can be screened for the presence of sequences homologous to the probe in a single experiment.