The HRT method is utilised for recombinants without introns. The most difficult step in applying hybridisation is obtaining a suitable probe. If the target DNA is represented with highly abundant mRNA, a small number of cDNA clones can be screened and the target gene can be identified by sequencing or hybrid released translation.
In HRT, the mRNA is hybridised to membrane bound cDNA. After washing with unbound mRNA, the bound mRNA is released and used in translation (in vitro). The translation products obtained are analysed by polyacrylamide gel electrophoresis. The cDNA clones can now be correlated with their protein product, and used as probes for isolating the recombinant target gene.
Immunochemical Detection
Immunochemical detection relies on the ability of the desired gene to produce its protein; however, in this method the presence of protein is detected by a specific antibody (and not by an assay for biological activity). There are many gene cloning procedures in which eukaryotic genes are identified in prokaryotic hosts by antibody recognition; while immunochemical detection involves challenging the fixed cells lysate with a primary antibody and then exposing to a labelled secondary antibody.
In this method, the primary antibody is bound to a polyvinyl sheet, on which the lysed, antigen-producing colonies or plaques are replica plated. The polyvinyl sheet is then exposed to labelled secondary antibody, which although is specific for the same antigen as the primary, but binds to different epitope. Colonies in which the target genes are produced are identified by autoradiography or by a colour change.
| Read More Topics |
| Nucleic acid hybridisation by replica plate method |
| Vectors for preparing single stranded DNA |
| Vectors for cloning larger DNA fragments |
