COMPOSITIONS AND METHODS FOR USE IN RECOMBINATIONAL CLONING OF NUCELIC ACIDS

• US 20110033920A1
• Filed: 12/29/2009
• Published: 02/10/2011
• Est. Priority Date: 03/02/1999
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Temple, Gary F., Hartley, James L., Brasch, Michael A., Cheo, David
• Granted Patent: US 8,241,896 B2
• Time in Patent Office: Days
• US Class Current: 435/320.1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 2800/70 Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites

Abstract

The present invention relates generally to compositions and methods for use in recombinational cloning of nucleic acid molecules. In particular, the invention relates to nucleic acid molecules encoding one or more recombination sites or portions thereof, to nucleic acid molecules comprising one or more of these recombination site nucleotide sequences and optionally comprising one or more additional physical or functional nucleotide sequences. The invention also relates to vectors comprising the nucleic acid molecules of the invention, to host cells comprising the vectors or nucleic acid molecules of the invention, to methods of producing polypeptides using the nucleic acid molecules of the invention, and to polypeptides encoded by these nucleic acid molecules or produced by the methods of the invention. The invention also relates to antibodies that bind to one or more polypeptides of the invention or epitopes thereof. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments.

Claims

1-38. -38. (canceled)

39. A nucleic acid segment, comprising, in order, the following components:

a) a first site-specific recombination site,

b) a ccdB gene,

c) a partial coding region of ccdA gene, and

d) a second site-specific recombination site.

40. The nucleic acid segment of claim 39, wherein the first or second site-specific recombination sites are att sites.

41. The nucleic acid segment of claim 39, wherein the first and second site-specific recombination sites do not recombine with each other.

42. The nucleic acid segment of claim 39, wherein the nucleic acid segment is a linear nucleic acid molecule.

43. The nucleic acid segment of claim 39, wherein the nucleic acid segment is included in a vector.

44. The nucleic acid segment of claim 39, wherein the nucleic acid segment additionally contains an antibiotic resistance marker between the first and second site-specific recombination sites.

45. A cloning vector comprising the following components:

a) a first site-specific recombination site,

b) a ccdB gene,

c) a partial coding region of ccdA gene, and

d) a second site-specific recombination site.

46. The cloning vector of claim 45 which is a plasmid.

47. The plasmid cloning vector of claim 46, further comprising contains a prokaryotic origin of replication and a eukaryotic promoter.

48. The plasmid cloning vector of claim 46, wherein the first or second site-specific recombination sites are att sites.

49. The plasmid cloning vector of claim 46, wherein the first and second site-specific recombination sites do not recombine with each other.