US5011769A
Expired / Lapsed
US
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Methods for detecting nucleic acid sequences
Assignee: ID Biomedical Corp of Quebec
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Filing: 1988-04-29
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Granted: 1991-04-30
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Expires: 2008-04-29
Abstract
Abstract This invention provides a method for detecting a target nucleic acid which comprises forming a reaction mixture which includes the target nucleic acid and an amount of a complementary single-stranded nucleic acid probe which is greater than the target molecule, under conditions which allow the probe and the target nucleic acid to hybridize to each other and form a double stranded target-probe complex, nicking the hybridized probe at least once within a predetermined sequence so as to form at least two probe fragments hybridized to the target nucleic acid, resulting in the probe fragments to become single-stranded and allowing the target nucleic acid to become hybridized to another probe; and identifying probe fragments, thereby detecting the target nucleic acid. This invention also provides a method for detecting a target nucleic acid.
Inventors
- 👤 Peter Duck
- 👤 Robert Bender
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 17:18
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682; C12Q1/6823; C12Q1/6827; C12Q1/6876
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
188 words
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 1 and NA 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.
Claim 2
Independent Claim
188 words
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 1 and NA 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.
Claim 3
Dependent Claim
26 words
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).
Claim 4
Dependent Claim
42 words
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).
Claim 5
Dependent Claim
15 words
A method of claim 1, wherein the scissile nucleic acid linkage is an RNA sequence.
Claim 6
Dependent Claim
28 words
A method of claim 1, wherein NA 1 and NA 2 independently comprise from 0 to about 20 deoxyribonucleotides and R comprises for 1 to about 100 ribonucleotides.
Claim 7
Dependent Claim
29 words
A method of claim 1, wherein n is an integer from 2 to 10 and wherein at least one of NA 1 or NA 2 varies within the probe.
Claim 8
Dependent Claim
9 words
A method of claim 1, wherein n is 1.
Claim 9
Dependent Claim
21 words
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.
Claim 10
Dependent Claim
11 words
A method of claim 8, wherein the ribonuclease is RNase H.
Claim 11
Dependent Claim
11 words
A method of claim 8, wherein the ribonuclease is Exo III.
Claim 12
Dependent Claim
14 words
A method of claim 1, wherein the probe is immobilized on a solid support.
Claim 13
Dependent Claim
27 words
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.
References Cited (Backward Citations)
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| 3.5 Years (Year 4) | 1994-04-29 | 1994-10-29 | 1995-04-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-04-29 | 1998-10-29 | 1999-04-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-04-29 | 2002-10-29 | 2003-04-30 | $7700.00 | Window Closed / Lapsed |