Cloning specific complete polyadenylylated 3'-terminal cDNA segments

Abstract

A simple method for extending partial cDNA clones of specific mRNA molecules is described. The method combines plasmid-primed first-strand cDNA synthesis [Okayama and Berg, Mol. Cell. Biol. 2 (1982) 161-170] with second-strand synthesis by primer extension. This approach selects for the synthesis of specific double-stranded cDNAs with complete 3' termini and poly(A) tails. We have used this method to extend preexisting cDNA clones to isolate 3' segments of human beta- and G gamma-globin cDNAs. These recombinants are particularly useful for the construction of templates for in vitro transcription of synthetic polyadenylylated mRNAs and pre-mRNAs. In addition, nucleotide sequence analysis of the cloned gamma-globin cDNA segment defined a previously unreported sequence polymorphism in the 3' untranslated region.