FEN endonucleases

• US 20060183207A1
• Filed: 10/27/2005
• Published: 08/17/2006
• Est. Priority Date: 01/24/1996
• Status: Abandoned Application

• Current Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Lyamichev, Victor I., Kaiser, Michael W., Lyamicheva, Natasha
• Application Number:
  US11/260,845
• Publication Number:
  US 20060183207A1
• Time in Patent Office: Days
• US Class Current: 435/183
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2561/109 Invader technology

Abstract

The present invention provides novel cleavage agents and polymerases for the cleavage and modification of nucleic acid. The cleavage agents and polymerases find use, for example, for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. In some embodiments, the 5′ nuclease activity of a variety of enzymes is used to cleave a target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

Claims

1. A composition comprising a purified recombinant FEN-1 endonuclease derived from Pyrococcus furious.

2. The composition of claim 1, further comprising nucleic acid molecules configured to form a cleavage structure cleavable by said FEN-1 endonuclease.

3. The composition of claim 2, wherein at least one of said nucleic acid molecules comprises a fluorescent label.

4. The composition of claim 1, further comprising a buffer comprising magnesium.

5. The composition of claim 1, wherein said purified recombinant FEN-1 endonuclease comprises SEQ ID NO:

115.

6. The composition of claim 1, wherein said purified recombinant FEN-1 endonuclease is encoded by an expression vector comprising SEQ ID NO:

114.

7. The composition of claim 1, further comprising a polymerase.

8. The composition of claim 2, wherein at least one of said nucleic acid molecules is generated by DNA replication.

9. A composition comprising a purified FEN-1 endonuclease derived from Pyrococcus furious.

10. The composition of claim 9, further comprising nucleic acid molecules configured to form a cleavage structure cleavable by said FEN-1 endonuclease.

11. The composition of claim 10, wherein at least one of said nucleic acid molecules comprises a fluorescent label.

12. The composition of claim 9, further comprising a buffer comprising magnesium.

13. The composition of claim 9, wherein said purified recombinant FEN-1 endonuclease comprises SEQ ID NO:

115.

14. The composition of claim 9, wherein said purified recombinant FEN-1 endonuclease is encoded by an expression vector comprising SEQ ID NO:

114.

15. The composition of claim 9, further comprising a polymerase.

16. The composition of claim 10, wherein at least one of said nucleic acid molecules is generated by DNA replication.

17. A composition comprising an expression vector encoding Pyrococcus furious FEN-1 endonuclease.

18. A method of cleaving a nucleic acid molecule comprising exposing a nucleic acid molecule to purified Pyrococcus furious FEN-1 endonuclease under conditions such that the nucleic acid is cleaved by the Pyrococcus furious FEN-1 endonuclease.

19. The method of claim 18, wherein said Pyrococcus furious FEN-1 endonuclease is heated to at least 55°

C. prior to said cleaving.

20. The method of claim 18, wherein said nucleic acid molecule comprises a label.

21. The method of claim 20, wherein said label comprises a fluorescent label.

22. The method of claim 18, wherein said nucleic acid molecule is hybridized to a target nucleic acid, wherein a second nucleic acid molecule is also hybridized to said target nucleic acid, wherein at least the 3′

terminal nucleotide of said second nucleic acid molecule overlaps with a region of the target nucleic acid that is complementary to said nucleic acid molecule.

23. The method of claim 22, wherein said 3′

terminal nucleotide is complementary to said target nucleic acid.

24. The method of claim 18, wherein said nucleic acid molecule is generated by DNA replication.