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.
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D007610:Kallikreins / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C033539:oligodeoxycytidylic acid; C033538:oligodeoxyguanylic acid; D004247:DNA; D007610:Kallikreins; D010196:Pancreatic Elastase