[44] Cloning of complementary DNA inserts from phage DNA directly into plasmid vector

The desired complementary DNA (cDNA) inserts are usually sub cloned from the positive phages into appropriate plasmid or M13 vectors for large-scale production. The cDNA insert isolated from the subcloning vector is then subjected to restriction enzyme mapping, southern blotting and hybridization, or nucleotide sequencing. Several methods have been designed to analyze the cDNA insert directly from the phage clone. Double-stranded h DNA can serve as a template for sequencing, using the chain termination method. The cDNA insert can be amplified for subcloning by the PCR. By incorporating the T7 RNA polymerase recognition sequence and the translation initiation codon into a primer, the amplification by the PCR has allowed subsequent in vitro transcription and translation of the phage cDNA inserts into protein. The method is likely to be extended to the cloning of inserts from other λ vectors when bacteria host contains pMC9— that is, (Y1088, Y1089, or YI090) or other plasmid vectors. More importantly, combination of this method with the PCR method, which allows the analysis of DNA directly from single bacterial colonies by boiling in H2O, made it practical to clone several cDNA inserts simultaneously.