A Method for Cloning Full-Length cDNA in Plasmid Vectors

The first step in the molecular analysis of many genetic systems is the isolation and cloning of a complementary deoxyribo nucleic acid (cDNA) copy of a messenger ribo nucleic acid (mRNA) expressed by the gene of interest. The sequence of full length cDNA clones allows one to deduce the amino acid sequence of the encoded protein. Full-length cDNA clones have been invaluable in the analysis of the organization and regulation of eukaryotic genes in many hybridization experiments. Various methods for the cloning of cDNA have been developed since the discovery of reverse transcriptase, the enzyme that made it all possible. For instance, comparison of full-length cDNA clones to the genes of these proteins allowed identifying the transcriptional start sites, translational signals in the leader sequence, and different usage of polyadenylation signals in the 3’ sequence of the mRNA. Various methods and vectors beside the one present in this chapter have been developed to cope with such a situation, among them are highly effective means of introducing the recombinant vectors into the bacterial host cell. Bacteriophage λ derived vectors allow in vitro packaging of the deoxyribo nucleic acid (DNA) and infection of the host.