A simple and very efficient method for generating cDNA libraries.
A simple method for generating cDNA libraries from submicrogram quantities of mRNA is described. It combines classical first-strand 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]. Neither 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 10(6) recombinants generated per microgram mRNA, a considerable improvement over earlier methods. Using the fully sequenced 1300 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.
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis*; D004745:Enkephalins / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D005914:Globins / Q000096:biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis; D012329:RNA, Bacterial / Q000096:biosynthesis
D004745:Enkephalins; D011498:Protein Precursors; D012329:RNA, Bacterial; D005914:Globins; D004247:DNA; C033660:preproenkephalin