Cloning specific complete polyadenylylated 3'-terminal cDNA segments.
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.
Lang
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Spritz
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D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005914:Globins / Q000235:genetics*; D006801:Humans; D011061:Poly A / Q000235:genetics*; D011110:Polymorphism, Genetic; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D005914:Globins; D004247:DNA