US 8,945,884 B2Granted

Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites

AssigneeLife Technologies Corporation

InventorsJonathan Chesnut, John Carrino, Louis Leong, Knut Madden, Martin Gleeson, James Fan, Michael Brasch, David Cheo, James Hartley, Devon Byrd, Gary Temple

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Abstract

The present invention provides compositions and methods for recombinational cloning. The compositions include vectors having multiple recombination sites and/or multiple topoisomerase recognition sites. The methods permit the simultaneous cloning of two or more different nucleic acid molecules. In some embodiments the molecules are fused together while in other embodiments the molecules are inserted into distinct sites in a vector. The invention also generally provides for linking or joining through recombination a number of molecules and/or compounds (e.g., chemical compounds, drugs, proteins or peptides, lipids, nucleic acids, carbohydrates, etc.) which may be the same or different. The invention also provides host cells comprising nucleic acid molecules of the invention or prepared according to the methods of the invention, and also provides kits comprising the compositions, host cells and nucleic acid molecules of the invention, which may be used to synthesize nucleic acid molecules according to the methods of the invention.

Claims12

Dependent claims

An in vitro method of cloning a nucleic acid molecule comprising:

(a) obtaining a first nucleic acid molecule to be cloned;
(b) mixing said first nucleic acid molecule to be cloned in vitro with a second nucleic acid molecule comprising at least a first topoisomerase recognition site flanked by at least a first recombination site, and at least a second topoisomerase recognition site flanked by at least a second recombination site, wherein said first and second recombination sites do not recombine with each other, and wherein said first and second recombination sites are selected from the group consisting of:
(a) attB sites,
(b) attP sites,
(c) attL sites,
(d) attR sites,
(e) lox sites,
(f) psi sites,
(g) dif sites,
(h) cer sites,
(i) frt sites;
and at least one topoisomerase;
and
(c) incubating said mixture under conditions such that said first nucleic acid molecule to be cloned is inserted into said second nucleic acid molecule between said first and second topoisomerase recognition sites, thereby producing a first product molecule comprising said first nucleic acid molecule to be cloned between said first and second recombination sites.

11 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/892,903Filed 13 May 2013 · continuation
Priority: 11 Dec 2000
Published: US 2013/0004996 A15 Dec 2013
Granted: 3 Feb 2015
Term ends: Not estimatedThe source records don’t support an estimate.

Classification14 codes

C12N 15/66
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for inserting a…
C12N 9/90
Enzymes; Proenzymes; Compositions thereof ; Processes for preparing, activating, inhibiting, separating or purifying enzymes: isomerases
C12N 15/10
Recombinant DNA-technology: processes for the isolation, preparation or purification of DNA or RNA
C12N 15/64
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for preparing…

Family

Application09/732,914Parent of this continuation-in-part · filed 11 Dec 2000
Application60/318,902Us provisional application · filed 14 Sep 2001
Application60/333,124Us provisional application · filed 27 Nov 2001
Application10/005,876Parent of this continuation-in-part · filed 7 Dec 2001
Application10/014,128Parent of this continuation-in-part · filed 7 Dec 2001
Application60/385,613Us provisional application · filed 5 Jun 2002
Application10/454,793Parent of this continuation · filed 5 Jun 2003
Application10/792,035Parent of this continuation · filed 4 Mar 2004
US 7,033,801 B2Parent of this continuation-in-part · filed 25 Apr 2006
Application11/612,445Parent of this continuation · filed 18 Dec 2006
US 7,198,924 B2Parent of this continuation · filed 3 Apr 2007
US 7,244,560 B2Parent of this continuation-in-part · filed 17 Jul 2007
US 7,393,632 B2Parent of this continuation-in-part · filed 1 Jul 2008
US 8,030,066 B2Parent of this continuation · filed 10 Apr 2011
Application13/219,440Parent of this continuation · filed 26 Aug 2011
US 2013/0323796 A1Pre-grant publication · published 5 Dec 2013
Application10/454,793Parent of this continuation-in-part
Application11/612,445Parent of this continuation-in-part
US 2002/0007051 A1Published application
US 2003/0186233 A1Published application
US 2004/0265863 A1Published application
US 2006/0029935 A1Published application
US 2008/0241889 A1Published application
US 2013/0004996 A1Published application
US 8,945,884 B2This patent

From the US filing history. Foreign family members aren’t listed.