Method and kit for detecting nucleic acid cleavage utilizing a covalently attached fluorescent tag

• US 5,786,139 A
• Filed: 12/09/1994
• Issued: 07/28/1998
• Est. Priority Date: 12/09/1994
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: PanVera Corp. (Vertex Pharmaceuticals Incorporated)
• Inventors: Burke, Thomas J., Bolger, Randall E., Checovich, William J., Thompson, David V.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Brusca, John S.
• Application Number:
  US08/353,079
• Time in Patent Office: 1,327 Days
• Field of Search: 435/6, 435/7.72, 435/196, 536/25.32
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation

Abstract

The present invention is based on the degradation of a fluorescence labeled nucleic acid such as DNA (F-DNA), RNA (F-RNA) or DNA/RNA substrate. The label may be covalently attached to the relatively high volume substrate at one position or many positions. The nucleic acid is then put in contact with another substance and activity is detected by Fluorescence Polarization.

Claims

1. A method of detecting cleavage of a nucleic acid comprising:

a) covalently attaching at least one fluorescence emitting label to a nucleotide of the nucleic acid to be cleaved;

b) measuring the fluorescence polarization of the label before step c);

c) incubating the labeled nucleic acid of step a) with a reagent to be tested wherein the reagent does not interact, in a manner that would diminish reagent activity enough to reduce useful sensitivity of cleavage measurement in step d), with the labeled nucleotide

d) measuring the fluorescence polarization of the label during step c); and

,

e) comparing the fluorescence polarization measurements of step b) with step d).

2. The method of claim 1 wherein the fluorescence emitting label is selected from the group consisting of fluorescein, fluorescein derivatives and fluorescein analogs.

3. The method of claim 1 wherein the fluorescence emitting label is attached to the nucleic acid with a 1 to 21 carbon chain.

4. The method of claim 1 wherein the nucleic acid consists of DNA.

5. The method of claim 4 wherein the DNA consists of an oligonucleotide of between 2 and 50 bases.

6. The method of claim 4 wherein the DNA consists of two complementary oligonucleotides of between 2 and 50 base pairs each.

7. The method of claim 3 wherein the fluorescence emitting label is attached to an end of the oligonucleotide.

8. The method of claim 1 wherein the nucleic acid consists of an RNA strand.

9. The method of claim 8 wherein the fluorescence emitting label is attached periodically along the RNA strand.

10. The method of claim 1 wherein the nucleic acid consists of a DNA/RNA hybrid.

11. The method of claim 1 wherein measuring the fluorescence polarization consists of quantitative detection of polarized light emitted from the fluorescence emitting label.

12. A method of detecting cleavage of a nucleic acid comprising:

a) covalently attaching a fluorescence emitting label to a nucleotide of the nucleic acid;

b) measuring the fluorescence polarization of the label before step c);

c) mixing a reagent to be tested with the labeled nucleic acid wherein the reagent does not interact, in a manner that would diminish reagent activity enough to reduce useful sensitivity of cleavage measurement in step e), with the labeled nucleotide

d) incubating the mixture of step c) for a time sufficient for the reagent to react with the nucleic acid;

e) measuring the fluorescence polarization of the label after step d); and

,

f) measuring the activity of the reagent by the degree of change in fluorescence polarization of the label.

13. The method of claim 12 wherein the fluoresence emitting label is from the group consisting of fluorescein, fluorescein derivatives and fluorescein analogs.

14. The method of claim 12 wherein cleavage is detected when the reagent of step (b) is a non-nuclease chemical substance.

15. The method of claim 12 wherein cleavage is detected when the reagent of step (b) is a nuclease.

16. The method of claim 12 wherein the label may be covalently attached to the reagent at one position or many positions.