Invitrogen Corporation

Life Technologies Corporation

Sloan-kettering Institute for Cancer Research

South Carolina Research Institute

John A Heyman

Knut R Madden

Stewart Shuman

Jonathan D Chesnut

Robert Bennett

Gabor Szalai

Michael Felder

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&#x2032; overhang at the same end, with a second ds nucleic acid molecule having a blunt end, such that the 5&#x2032; 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.

Methods and reagents for molecular cloning

A Vaccinia topoisomerase-adapted linker, which contains an oligonucleotide primer binding site of known sequence, useful for specifically joining the linker to the end of a polynucleotide of unknown sequence and allowing subsequent PCR amplification of the polynucleotide or DNA is provided. Kits containing the invention Vaccinia topoisomerase-adapted linker and one or more linker-specific oligonucleotides for annealing to the linker in PCR are also provided. In addition, the invention provides methods for using the invention linkers in linker-mediated PCR amplification procedures for isolation and optional sequencing of isolated PCR amplification products.

Topoisomerase linker-mediated amplification methods

Growing community of inventors

Rixensart, Belgium

John A Heyman