Continous fluorometric assay for detecting nucleic acid cleavage

• US 5,763,181 A
• Filed: 08/30/1996
• Issued: 06/09/1998
• Est. Priority Date: 12/30/1994
• Status: Expired due to Term

• Current Assignee: Georgetown University
• Original Assignee: Georgetown University
• Inventors: Lee, S. Paul, Han, Myun Ki, Chirikjian, Jack G.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Fredman, Jeffrey
• Application Number:
  US08/706,135
• Time in Patent Office: 648 Days
• Field of Search: 435/6, 435/18, 435/91.2, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/23.1
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

A method of detecting an enzyme-mediated DNA cleavage reaction in a fluorometric assay is provided. The method can be used to detect DNA cleavage caused by restriction endonucleases, retroviral integrase enzymes, DNases, RNases, or enzymes utilized in other strand separating processes in molecular biology.

Claims

1. A method of detecting continuously an endonuclease-mediated nucleic acid cleavage reaction in a fluorometric assay comprising the steps of:

(a) preparing a fluorescently labeled oligonucleotide containing a double-stranded nucleic acid sequence recognizable by said endonuclease, wherein said oligonucleotide acts as a substrate for said endonuclease;

(b) contacting said oligonucleotide of step (a) with said endonuclease in an amount sufficient to cleave said oligonucleotide to produce oligonucleotide products; and

(c) detecting continuously a nucleic acid cleavage reaction by detecting an increase in fluorescence intensity.

2. The method of claim 1, wherein said oligonucleotide is fluorescently labeled at one end.

3. The method of claim 1, wherein said oligonucleotide is fluorescently labeled at both ends.

4. The method of claim 1, wherein said nucleic acid is DNA.

5. The method of claim 1, wherein said endonuclease is a restriction endonuclease.

6. The method of claim 1, wherein said endonuclease is a DNase or RNase enzyme.

7. The method of claim 1, wherein said endonuclease is a retroviral integrase enzyme.

8. The method of claim 2, wherein said endonuclease is a restriction endonuclease.

9. The method of claim 3, wherein said endonuclease is a retroviral integrase enzyme.

10. The method of claim 5, wherein said restriction endonuclease is BamHI.

11. The method of claim 7, wherein said retroviral integrase enzyme is HIV integrase.

12. The method of claim 1, wherein said endonuclease-mediated nucleic acid cleavage reaction occurs during a process for amplifying or detecting a specific DNA or RNA sequence.

13. The method of claim 12, wherein said process for amplifying or detecting a DNA or RNA sequence is catalytic hybridization amplification.

14. The method of claim 13, wherein said endonuclease is RNase H.

15. The method of claim 14, wherein said oligonucleotide is fluorescently labeled at both ends.