Bacteriophage λ Cloning System for the Construction of Directional cDNA Libraries

We have developed a bacteriophage λ cloning vector, λ ORF8, that can be used for the construction of cDNA libraries. The wild-type λ genome contains five BamHI, five EcoRI, and seven HindIII restriction sites that have all been removed from the genome of λ ORF8. Sites for these endonucleases are present within the multiple cloning site of λ 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, will create a HindIII 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 λ ORF8. To demonstrate the utility of this cloning system, we prepared a library from 5 μ g of mRNA isolated from phytohemagglutinin-stimulated human peripheral blood lymphocytes. The primary library contained 2 × 108 plaque-forming phage, at least 80% of which contain inserts. A portion of the library was examined for the presence of γ -interferon-related 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.