Compositions for use in recombinational cloning of nucleic acids

• US 7,670,823 B1
• Filed: 03/02/2000
• Issued: 03/02/2010
• Est. Priority Date: 03/02/1999
• Status: Expired due to Fees

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated), 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
• Primary Examiner(s): Johannsen; Diana B
• Application Number:
  US09/517,466
• Time in Patent Office: 3,652 Days
• Field of Search: 536/23.1, 536/24.1, 536/24.33, 435/252.3, 435/320.1, 435/325, 435/810
• US Class Current: 435/252.3
• 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. An isolated nucleic acid molecule comprising a lambda att recombination site wherein a sequence of the seven base pair overlap region within the fifteen base pair core region is ATTATAC.

2. The isolated nucleic acid molecule of claim 1, which comprises two or more lambda att recombination sites.

3. The isolated nucleic acid molecule of claim 1, wherein the lambda att-recombination site is located between a transcriptional regulatory sequence and an open reading frame, wherein the transcriptional regulatory sequence and open reading frame are operably linked.

4. The isolated nucleic acid molecule of claim 3, wherein the transcriptional regulatory sequence is a promoter.

5. The isolated nucleic acid molecule of claim 1, which is a vector.

6. The isolated nucleic acid molecule of claim 5, wherein the vector is a plasmid.

7. The isolated nucleic acid molecule of claim 1, which further comprises at least one origin of replication.

8. The isolated nucleic acid molecule of claim 7, which comprises at least two origins of replication.

9. The isolated nucleic acid molecule of claim 7, which comprises at least one origin of replication that allows for replication of the nucleic acid molecule in a prokaryotic cell.

10. The isolated nucleic acid molecule of claim 9, wherein the prokaryotic cell is an Escherichia coli cell.

11. An isolated host cell comprising one or more isolated nucleic acid molecules of claim 1.