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

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.