A simple and very efficient method for generating cDNA libraries

Abstract

A simple method for generating cDNA libraries from submicrogram quantities of mRNA is describe. It combines classical first-stand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis [Okayama, H., and Berg, P., Mol. Cell. Biol. 2 (1982) 161–170]. Neiher the elaborate vector-primer system nor the classical hairpin loop cleavage by S1 nuclease are used. cDNA thus made can be tailed and cloned without further purification or sizing. Cloning efficiencies can be as high as 10⁶ recombinants generated per μg mRNA, a considerable improvement over earlier methods. Using the fully sequenced1300 nucleotide-long bovine preproenkephalin mRNA, we have established by sequencing that the method yields faithful full-length transcripts. This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.