Enzymes for the detection of specific nucleic acid sequences

• US 6,759,226 B1
• Filed: 05/24/2000
• Issued: 07/06/2004
• Est. Priority Date: 05/24/2000
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Lyamicheva, Natalie E., Schaefer, James J., Kaiser, Michael W., Allawi, Hatim Taysir, Ma, Wu-Po, Neri, Bruce P., Lyamichev, Victor I.
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US09/577,304
• Time in Patent Office: 1,504 Days
• Field of Search: 435/199, 435/471, 435/91.1, 435/975
• US Class Current: 435/199
• 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 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to novel enzymes designed for direct detection, characterization and quantitation of nucleic acids, particularly RNA. The present invention provides enzymes that recognize specific nucleic acid cleavage structures formed on a target RNA sequence and that cleave the nucleic acid cleavage structure in a site-specific manner to produce non-target cleavage products. The present invention provides enzymes having an improved ability to specifically cleave a DNA member of a complex comprising DNA and RNA nucleic acid strands.

Claims

1. A composition comprising a polymerase comprising a 5′

nuclease, wherein said polymerase comprises a heterologous functional domain, wherein said heterologous functional domain comprises an amino acid sequence that provides improved background specificity in a nucleic acid cleavage assay compared to said polymerase enzyme without said heterologous functional domain.

2. The composition of claim 1, wherein said 5′

nuclease comprises a thermostable 5′

nuclease.

3. The composition of claim 1, wherein said polymerase is altered in sequence relative to a naturally occurring sequence of a polymerase such that it exhibits reduced DNA synthetic activity from that of the naturally occurring polymerase.

4. The composition of claim 1, wherein said polymerase comprises a thermostable polymerase.

5. The composition of claim 4, wherein said thermostable polymerase comprises a polymerase from a Thermus species.

6. The composition of claim 5 wherein said Thermus species is selected from the group consisting of Thermus aquaticus, Thermus flavus, Thermus thermophilus, Thermus filiformus, and Thermus scotoductus.

7. The composition of claim 1, wherein said heterologous functional domain comprises an amino acid sequence that provides an improved substrate binding activity in said nucleic acid cleavage assay.

8. The composition of claim 1, wherein said heterologous functional domain comprises two or more amino acids from a polymerase domain of a second polymerase.

9. The composition of claim 8, wherein at least one of said two or more amino acids is from a palm region of said polymerase domain of said second polymerase.

10. The composition of claim 8, wherein at least one of said two or more amino acids is from a thumb region of said polymerase domain of said second polymerase.

11. The composition of claim 8, wherein said second polymerase comprises Thermus thermophilus polymerase.

12. The composition of claim 1, wherein said nucleic acid cleavage assay comprises cleavage of a DNA member of a substrate containing at least one RNA component.

13. The composition of claim 1, wherein said nucleic acid cleavage assay comprises an invasive cleavage assay.

14. A kit comprising the composition of claim 1.

15. The kit of claim 14, further comprising at least one nucleic acid cleavage substrate.

16. The kit of claim 15, further comprising at least one RNA capable of hybridizing to said nucleic acid cleavage substrate.

17. The kit of claim 14, further comprising a labeled oligonucleotide.

18. The kit of claim 14, further comprising an invasive oligonucleotide.

19. A method for cleaving a nucleic acid comprising:

a) providing;

i) the composition of claim 1; and

ii) a substrate nucleic acid; and

b) exposing said substrate nucleic acid to said enzyme.

20. The method of claim 19, wherein said exposing said substrate nucleic acid to said enzyme produces at least one cleavage product.

21. The method of claim 20, further comprising the step of c) detecting said cleavage product.