Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors

Abstract

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.