Construction of uni-directionally cloned cDNA libraries from messenger RNA for improved 3′ end DNA sequencing

US6864057B2 · kind B2 · utility

Assignee

• Incyte Corporation · US

Inventors

• Glenn K. Fu · Dublin, US
• Steven Starnes · East Palo Alto, US
• Laura Stuve · Los Gatos, US

Key dates

• Filing date: Jan 4, 2002
• Grant date: Mar 8, 2005
• Expiry date: Sep 25, 2022

Classification

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

Abstract

Methods are provide for preparing cDNA corresponding to a mRNA. In the subject methods, a mRNA is first contacted with a mixture of primers under first strand cDNA synthesis conditions. The primer mixture contains primers that have at least 10 contiguous deoxythymidines, a double stranded restriction enzyme recognition sequence near one end and a non-polyA-complementary region near the other end, where the non-polyA-complementary region is -VV, -VTV, -VTTV, -VTTTV, and -VVVVV. The resultant cDNA is modified such that the polyT tail is substantially removed. The modified cDNA is then ligated into a vector. The subject methods find use in a variety of applications, and find particular use in the sequencing of DNA and in the synthesis of cDNA libraries.