Methods and reagents for molecular cloning

US6916632B2 · kind B2 · utility

Assignees

• Invitrogen Corporation · US
• Sloan Kettering Institute for Cancer Research · US

Inventors

• Jonathan Chesnut · Carlsbad, US
• Stewart Shuman · New York, US
• Knut Madden · Carlsbad, US
• John Heyman · Delano, US
• Robert Bennett · Bound Brook, US

Key dates

• Filing date: Aug 21, 2001
• Grant date: Jul 12, 2005
• Expiry date: May 19, 2022

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/64
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The present invention provides compositions, methods, and kits for covalently linking nucleic acid molecules. The methods include a strand invasion step, and the compositions and kits are useful for performing such methods. For example, a method of covalently linking double stranded (ds) nucleic acid molecules can include contacting a first ds nucleic acid molecule, which has a topoisomerase linked to a 3′ terminus of one end and has a single stranded 5′ overhang at the same end, with a second ds nucleic acid molecule having a blunt end, such that the 5′ overhang can hybridize to a complementary sequence of the blunt end of the second nucleic acid molecule, and the topoisomerase can covalently link the ds nucleic acid molecules. The methods are simpler and more efficient than previous methods for covalently linking nucleic acid sequences, and the compositions and kits facilitate practicing the methods, including methods of directionally linking two or more ds nucleic acid molecules.