TY - JOUR
T1 - Cloning cDNAs corresponding to the 5′ end of messenger RNAs using specific DNA primers
AU - Dagorn, Jean Charles
AU - MacDonald, Raymond J.
N1 - Funding Information:
We thank Dr. Galvin Swift for helpful suggestions throughout this work and for a critical reading of the manuscript, and Marie Rotondi for preparing the manuscript. This work was supported by Grant AM27430 from the National Institutes of Health. J.-C.D. was a recipient of a Fogarty International Center Research Fellowship.
PY - 1984
Y1 - 1984
N2 - 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.
AB - 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.
KW - cloning
KW - mRNA
KW - primer-extension
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U2 - 10.1016/0300-9084(84)90256-6
DO - 10.1016/0300-9084(84)90256-6
M3 - Article
C2 - 6570693
AN - SCOPUS:0021525563
SN - 0300-9084
VL - 66
SP - 673
EP - 679
JO - Biochimie
JF - Biochimie
IS - 11-12
ER -