Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end

US5962271A · kind A · utility

Assignee

• Cloutech Laboratories, Inc. · US

Inventors

• Alex Chenchik · Redwood City, US
• York Yuan Yuan Zhu · Palo Alto, US
• Luda Diatchenko · Palo Alto, US
• Paul J. Siebert · Sunnyvale, US

Key dates

• Filing date: Jan 3, 1996
• Grant date: Oct 5, 1999
• Expiry date: Jan 3, 2016

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/1096
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

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.