Abstract
We have modified current methods to create a very efficient technique for cloning cDNAs in a defined orientation, into plasmid vectors bearing phage SP6 and T7 polymerase promoters. First strand synthesis is primed at the poly(A) tail with a 26-mer synthetic oligonucleotide linker/primer, the RNA is hydrolyzed and the cDNA is tailed with 10 to 15 dG residues. The cDNA is then annealed to two prepared vector fragments specific for the two ends of the cDNA (one bearing a dC10-15 tail and the other bearing a 14-nucleotide cohesive end complementary to the linker/primer). After ligation the second strand is synthesized with the large fragment of DNA polymerase I. Libraries of up to 8 x 10(6) independent transformants have been obtained from 1 microgram of Drosophila poly(A)+ RNA. The design of the method and careful optimization of first strand synthesis have permitted cloning of several large (4.3 to 6.5 kb), low abundance cDNAs. Transcription of essentially full-length clones with phage SP6 RNA polymerase produces RNAs that are efficiently translated in vitro to give complete, unfused products, thus permitting rapid characterization of the clones via the encoded polypeptides. Antisense RNAs can also be produced by transcription with phage T7 RNA polymerase.
