Bacteriophage lambda cloning system for the construction of directional cDNA libraries.

Publisher Website
Google Scholar
Abstract
We have developed a bacteriophage .lambda. cloning vector, .kappa.ORF8, that can be used for the construction of cDNA libraries. The wild-type .lambda. genome contains five BamHI, five EcoRI, and seven HindIII restriction sites that have all been removed from the genome of .lambda.ORF8. Sites for these endonucleases are present within the multiple cloning site of .lambda.ORF8. We report a method for preparing cDNAs that can be cloned in a single orientation in our phage vector. The method utilizes the synthesis of double-stranded cDNA, including priming of first-strand synthesis by oligo(dT). After completion of second-strand synthesis, a bifunctional oligodeoxynucleotide linker is ligated to the cDNA fragments. This linker, which contains a BamHI restriction site when ligated to the 3'' end of cDNA fragments. Subsequent treatment of methylated cDNA with HindIII and BamHI endonucleases allows these fragments to be cloned directionally into .lambda.ORF8. To demonstrate the ability of this cloning system, we prepared a library from 5 .mu.g of mRNA isolated from phytohemagglutinin-stimulated human peripheral blood lymphocytes. The primary library contained 2 .times. 108 plaque-forming phage, at least 80% of which contain inserts. A portion of the library was examined for the presence of .gamma.-interferon-related clones to verify the method had generated a library that was representative of phytohemagglutinin-stimulated peripheral blood lymphocytes. This simple and efficient cDNA cloning system significantly reduces the amount of RNA and effort required for the preparation of large directionally cloned libraries.