Identifying Clones

Bacterial clones containing the sought after recombinant vectors can be identified by hybridisation with specific radioactively labelled or enzyme-labelled cDNA or genomic DNA probes or alternatively by the immunodetection of protein products (using specialised expression vectors which allow a cloned foreign cDNA to be transcribed to express its protein product). Both approaches are technically straightforward. Both involve the transfer of bacterial colonies from a master agar plate on to carefully orientated nitrocellulose or nylon membranes. The cells are then lysed and the DNA (or protein) from the lysed colonies is immobilised on the membrane, which is used for the probing step. Recombinant colonies can be detected as spots on X-ray film either by autoradiography or enzyme-generated chemiluminescence. The spots on the X-ray film can then be aligned with the agar master plate allowing the correct colonies to be picked. For protein detection in expression vectors, antibody probes are employed and in a manner analogous to an ELISA test. The antibody probe is conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase. It is the activity of the bound enzyme on its chemiluminescence or chromogenic substrate that reveals the position of the recombinant colonies.