Claims
- 1. A method for detecting a single-stranded target nucleic acid which comprises:
- (a) obtaining the single-stranded target nucleic acid;
- (b) forming a reaction mixture which includes the target nucleic acid and a complementary single-stranded nucleic acid probe under conditions which allow the target nucleic acid and the probe to hybridize to each other and form a double-stranded, target-probe complex, the probe being present in molar excess relative to the target and having the structure
- [NA.sub.1 - R - NA.sub.2 ].sub.n
- wherein NA.sub.1 and NA.sub.2 are DNA sequences, wherein R is a scissile nucleic acid linkage, and wherein n is an integer from 1 to 10;
- (c) treating the double-stranded, target-probe complex from step (b) so as to cleave the probe within a predetermined sequence of the scissile nucleic acid linkage and thereby form at least one intact DNA-containing oligonucleotide fragment from the probe, such fragment being, or being treated so as to be, no longer capable of remaining hybridized to the target nucleic acid;
- (d) repeating steps (b) and (c); and
- (e) detecting the intact DNA-containing fragments so formed and thereby detecting the single-stranded target nucleic acid.
- 2. A method of claim 1, wherein the oligonucleotide fragment in step (c) is labelled with a detectable marker and labelled fragments are detected in step (e).
- 3. A method of claim 1, wherein the oligonucleotide fragment in step (c) is unlabelled, but capable of being labelled with a detectable marker, the fragment is so labelled prior to step (d) or (e), and labelled fragments are detected in step (e).
- 4. A method of claim 1, wherein the scissile nucleic acid linkage is an RNA sequence.
- 5. A method of claim 1, wherein NA.sub.1 and NA.sub.2 independently comprise from 0 to about 20 deoxyribonucleotides and R comprises for 1 to about 100 ribonucleotides.
- 6. A method of claim 1, wherein n is an integer from 2 to 10 and wherein at least one of NA.sub.1 or NA.sub.2 varies within the probe.
- 7. A method of claim 1, wherein n is 1.
- 8. A method of claim 1, wherein the treating in step (c) comprises contacting the double-stranded target-probe complex with a double-strand-specific ribonucluclease.
- 9. A method of claim 8, wherein the ribonuclease is RNase H.
- 10. A method of claim 8, wherein the ribonuclease is Exo III.
- 11. A method of claim 1, wherein the probe is immobilized on a solid support.
- 12. A method of claim 3, wherein the unlabelled fragment has a 3'-hydroxyl group and wherein the labelling of the fragment comprises RNA tailing from the 3'-hydroxyl group.
Parent Case Info
This application is a continuation-in-part of U.S. Application Ser. No. 805,279, filed Dec. 5, 1985, U.S. Pat. No. 4,876,187, the contents of which are hereby incorporated by reference.
US Referenced Citations (2)
Foreign Referenced Citations (1)
Number |
Date |
Country |
0227976 |
Aug 1987 |
EPX |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
805279 |
Dec 1985 |
|