Cloning cDNAs corresponding to the 5′ end of messenger RNAs using specific DNA primers

Jean Charles Dagorn, Raymond J. MacDonald

Research output: Contribution to journalArticle

Abstract

A method is described to clone cDNAs corresponding to the 5′ end of specific mRNAs which are absent from cDNA libraries. A segment is excised from the 5′ end of a previously cloned truncated cDNA and hybridized to total polyadenylated RNA. The hybridized primer is extended using reverse transcriptase, tailed with deoxycytidine, and the second strand synthesized using oligo (dG)10-16 as a primer. The primer-extended double-stranded cDNA is cloned into pBR322 by dC-dG homopolymeric tailing. This method has been successful for cloning a 398 bp cDNA fragment corresponding to the entire 5′ end of rat pancreatic elastase I mRNA, a prominent pancreatic mRNA, and a 510 bp fragment encoding the 5′ end of kallikrein mRNA, a lower abundant pancreatic mRNA.

Original languageEnglish (US)
Pages (from-to)673-679
Number of pages7
JournalBiochimie
Volume66
Issue number11-12
DOIs
StatePublished - Jan 1 1984

Keywords

  • cloning
  • mRNA
  • primer-extension

ASJC Scopus subject areas

  • Biochemistry

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