Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-01-03
1999-10-05
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 912, 536 231, 536 242, 536 253, C12P 1934, C07H 2104, C07H 2102
Patent
active
059622725
ABSTRACT:
The present invention pertains to methods for the synthesis and cloning of full-length cDNA, or cDNA fragments, that correspond to the complete sequence of 5'-ends of mRNA molecules. The method of the present invention comprises contacting RNA with a cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA-cDNA hybrid. The template switching oligonucleotide hybridizes to the CAP site at the 5'-end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3'-end of the ss cDNA that is complementary to the template switching oligonucleotide. The resulting full-length ss cDNA includes the complete 5'-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA.
The subject invention also pertains to the template switching oligonucleotides that can be used according to the subject method. Kits containing the template switching oligonucleotide are also included within the scope of the present invention.
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Chenchik Alex
Diatchenko Luda
Siebert Paul
Zhu York
Adler Benjamin Aaron
Clontech Laboratories, Inc.
Horlick Kenneth R.
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