Clontech Laboratories, Inc.

Cloutech Laboratories, Inc.

York Zhu

Alex Chenchik

Paul Siebert

Luda Diatchenko

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.

Methods and compositions for full-length cDNA Cloning using a

Described are compositions and methods which allow for the efficient addition of a defined sequence at the 3'-end of a full-length cDNA in the course of first-strand cDNA synthesis from an mRNA template. A cDNA synthesis primer that is capable of annealing to mRNA is used to prime the first strand synthesis reaction. An oligonucleotide that is linked to the 5'-end of the mRNA serves as a short, extended template such that when the reverse transcriptase enzyme reaches the 5'-end of the mRNA, the enzyme switches templates and proceeds to transcribe through the end of the linked oligonucleotide. As a result, the single-stranded cDNA product which corresponds to the full-length mRNA, will have at the 3'-end a defined sequence which is complementary to the linked oligonucleotide. A conservative element in the oligonucleotide sequence responsible for this reaction can include 3 to 5 guanylic acid residues at the 3'-end of the oligonucleotide. The subject invention provides for the increased synthesis of full-length cDNA from mRNA templates. The full-length cDNA prepared according to the present invention can then be amplified using PCR or cloned using standard procedures.

Growing community of inventors

Sunnyvale, CA, United States of America

York Zhu