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Children's Medical Center Corporation

Stratagene California

Stratagene

Stratagene, La Jolla, California

John C Bauer

Jeffrey Carl Braman

Raif S Geha

Dowain A Wright

Joseph A Sorge

Alan L Greener

Carsten-Peter Carstens

Henry Ji

Richard Gibbs

A shutter assembly includes a main body having a first side member and a second side member. Each of the first side member and the second side member has a first attachment feature. The shutter assembly also includes at least one cap. The cap has a pair of spaced apart second attachment features. The first attachment features of the first side member and the second side member of the main body and the second attachment features of the cap cooperate with one another to selectively secure the cap to the main body.

Shutter

The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

Circular site-directed mutagenesis

The invention provides methods of covalently joining nucleic acid molecules and methods of molecular cloning. The methods provide either sequential or simultaneous ligation of flanking or vector nucleic acid molecules to nucleic acid insert molecules by topoisomerase and DNA ligase. The methods provide for directional and non-directional covalent joining and cloning of nucleic acid molecules.

Method for ligating nucleic acids and molecular cloning

Growing community of inventors

San Diego, CA, United States of America

John C Bauer