US4725537A
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Assay, reagent and kit employing nucleic acid strand displacement and restriction endonuclease cleavage
Assignee: Genetics Institute LLC
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Filing: 1985-09-19
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Granted: 1988-02-16
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Expires: 2005-09-19
Abstract
Abstract A target nucleotide sequence having a half-restriction site is determined in the nucleic acid of a biological sample. It is contacted with a reagent complex of: (i) a labeled probe polynucleotide containing a target binding region which is substantially complementary to the target nucleotide sequence and which contains a unique half-restriction site completely complementary to the half-restriction site of the target nucleotide sequence; and (ii) a second polynucleotide hybridized to the labeled probe polynucleotide in at least a portion of the target binding region, the portion including the unique half-restriction site of the target binding region; The second polynucleotide contains at least one mismatched or unpaired nucleotide opposite to the unique half-restriction site of the labeled probe polynucleotide, whereby a restriction enzyme specific for the unique restriction site will not cleave the reagent complex. The target nucleotide sequence, if present, displaces the second polynucleotide from the target nucleotide region and forms the unique restriction site in double-stranded form. The labeled probe polynucleotide is then cleaved at the unique restriction site in double-stranded form to form labeled cleavage fragment, which is detected.
Inventors
- 👤 Edward F. Fritsch
- 👤 Jon I. Williams
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 20:50
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6823; C12Q1/6827; C12Q1/683; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
203 words
A method for determining a target nucleotide sequence having a half-restriction site in the nucleic acid of a biological sample which comprises the steps: (a) providing a reagent complex of: (i) a labeled probe polynucleotide containing a target binding region which is substantially complementary to the target nucleotide sequence and which contains a unique half-restriction site completely complementary to the half-restriction site of the target nucleotide sequence; and (ii) a second polynucleotide hybridized to the labeled probe polynucleotide in at least a portion of the target binding region the portion including the unique half-restriction site of the target binding region; the second polynucleotide containing at least one mismatched or unpaired nucleotide opposite to the unique half-restriction site of the labeled probe polynucleotide, whereby a restriction enzyme specific for the unique restriction site will not cleave the reagent complex; (b) contacting the reagent complex with the sample under conditions in which the target nucleotide sequence, if present, will displace the second polynucleotide from the target binding region and form the unique restriction site in double-stranded form; (c) specifically cleaving the labeled probe polynucleotide at the unique restriction site in double-stranded form to form a labeled cleavage fragment; and (d) detecting the labeled cleavage fragment.
Claim 2
Independent Claim
203 words
A method for determining a target nucleotide sequence having a half-restriction site in the nucleic acid of a biological sample which comprises the steps: (a) providing a reagent complex of: (i) a labeled probe polynucleotide containing a target binding region which is substantially complementary to the target nucleotide sequence and which contains a unique half-restriction site completely complementary to the half-restriction site of the target nucleotide sequence; and (ii) a second polynucleotide hybridized to the labeled probe polynucleotide in at least a portion of the target binding region the portion including the unique half-restriction site of the target binding region; the second polynucleotide containing at least one mismatched or unpaired nucleotide opposite to the unique half-restriction site of the labeled probe polynucleotide, whereby a restriction enzyme specific for the unique restriction site will not cleave the reagent complex; (b) contacting the reagent complex with the sample under conditions in which the target nucleotide sequence, if present, will displace the second polynucleotide from the target binding region and form the unique restriction site in double-stranded form; (c) specifically cleaving the labeled probe polynucleotide at the unique restriction site in double-stranded form to form a labeled cleavage fragment; and (d) detecting the labeled cleavage fragment.
Claim 3
Dependent Claim
30 words
The method of claim 1 wherein the labeled cleavage fragment is separated from uncleaved labeled probe polynucleotides after the specifically cleaving step (c) and prior to the detecting step (d).
Claim 4
Dependent Claim
20 words
The method of claim 2 wherein the labeled probe polynucleotide in the reagent complex is immobilized to a solid support.
Claim 5
Dependent Claim
45 words
The method of claim 3 wherein the labeled probe polynucleotide is immobilized adjacent to one of its ends and contains a detectable tag at or adjacent to the opposite end, and the unique half-restriction site is between the point of immobilization and the detectable tag.
Claim 6
Dependent Claim
19 words
The method of claim 4 wherein the second polynucleotide is hybridized to a portion of the target binding region.
Claim 7
Dependent Claim
23 words
The method of claim 5 wherein the second polynucleotide is hybridized to a portion adjacent to one end of the target binding region.
Claim 8
Dependent Claim
25 words
The method of claim 6 wherein the unique half-restriction site is intermediate within the portion of the target binding region hybridized to the second polynucleotide.
Claim 9
Dependent Claim
27 words
The method of claim 2 wherein the second polynucleotide is hybridized to a portion of the probe polynucleotide adjacent to one end of the target binding region.
Claim 10
Dependent Claim
25 words
The method of claim 8 wherein the unique restriction site is intermediate within the portion of the target binding region hybridized to the second polynucleotide.
Claim 11
Dependent Claim
23 words
The method of claim 1 wherein the second polynucleotide is hybridized to a portion adjacent to one end of the target binding region.
Claim 12
Dependent Claim
27 words
The method of claim 1 wherein the second polynucleotide contains a second detectable tag and the method further comprises the step: (e) further detecting displaced second polynucleotide.
Claim 13
Dependent Claim
35 words
The method of claim 11 further comprising the step: (f) comparing the quantity of detected labeled cleavage fragment from detecting step (d) with the quantity of detected displaced second polynucleotide from further detecting step (e).
Claim 14
Dependent Claim
130 words
A reagent complex for determining a target nucleotide sequence having a half-restriction site in the nucleic acid of a biological sample, comprising: (i) a labeled probe polynucleotide containing a target binding region which is substantially complementary to the target nucleotide sequence and which forms a unique half-restriction site complementary to the half-restriction site of the target nucleotide sequence; and (ii) a second polynucleotide hybridized to the labeled probe polynucleotide in at least a portion of the target binding region, the portion including the unique half-restriction site of the target binding region; the second polynucleotide containing at least one mismatched nucleotide opposite to the unique half-restriction site of the labeled probe polynucleotide, whereby a restriction enzyme specific for the unique restriction site in double-stranded form will not cleave the reagent complex.
Claim 15
Dependent Claim
130 words
A reagent complex for determining a target nucleotide sequence having a half-restriction site in the nucleic acid of a biological sample, comprising: (i) a labeled probe polynucleotide containing a target binding region which is substantially complementary to the target nucleotide sequence and which forms a unique half-restriction site complementary to the half-restriction site of the target nucleotide sequence; and (ii) a second polynucleotide hybridized to the labeled probe polynucleotide in at least a portion of the target binding region, the portion including the unique half-restriction site of the target binding region; the second polynucleotide containing at least one mismatched nucleotide opposite to the unique half-restriction site of the labeled probe polynucleotide, whereby a restriction enzyme specific for the unique restriction site in double-stranded form will not cleave the reagent complex.
Claim 16
Dependent Claim
16 words
The reagent complex of claim 13 wherein the labeled probe polynucleotide is immobilized to a support.
Claim 17
Dependent Claim
42 words
The reagent complex of claim 14 wherein the labeled probe polynucleotide is immobilized adjacent to one end and contains a detectable tag adjacent to the opposite end, and the unique half-restriction site is between the point of immobilization and the detectable tag.
Claim 18
Dependent Claim
20 words
The reagent complex of claim 15 wherein the second polynucleotide is hybridized to a portion of the target binding region.
Claim 19
Dependent Claim
27 words
The reagent of claim 16 wherein the second polynucleotide is hybridized to a portion of the probe polynucleotide adjacent to one end of the target binding region.
Claim 20
Dependent Claim
26 words
The reagent complex of claim 17 wherein the unique half-restriction site is intermediate within the portion of the target binding region hybridized to the second polynucleotide.
Claim 21
Dependent Claim
29 words
The reagent complex of claim 14 wherein the second polynucleotide is hybridized to a portion of the labeled probe polynucleotide adjacent to one end of the target binding region.
Claim 22
Dependent Claim
24 words
The reagent complex of claim 13 wherein the second polynucleotide is hybridized to a portion adjacent to one end of the target binding region.
Claim 23
Dependent Claim
26 words
The reagent complex of claim 20 wherein the unique half-restriction site is intermediate within the portion of the target binding region hybridized to the second polynucleotide.
Claim 24
Dependent Claim
15 words
The reagent complex of claim 13 wherein the second polynucleotide contains a second detectable tag.
Claim 25
Dependent Claim
33 words
A diagnostic kit comprising the reagent complex of claim 22 and a restriction endonuclease capable of specifically cleaving the unique half-restriction enzyme recognition site when it is in a perfectly matched double-stranded form.
Claim 26
Dependent Claim
33 words
A diagnostic kit comprising the reagent complex of claim 14 and a restriction endonuclease capable of specifically cleaving the unique half-restriction enzyme recognition site when it is in a perfectly matched double-stranded form.
Claim 27
Dependent Claim
33 words
A diagnostic kit comprising the reagent complex of claim 13 and a restriction endonuclease capable of specifically cleaving the unique half-restriction enzyme recognition site when it is in a perfectly matched double-stranded form.
References Cited (Backward Citations)
EP0142299A2
Cited
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1991-02-15 | 1991-08-17 | 1992-02-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-02-15 | 1995-08-17 | 1996-02-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-02-15 | 1999-08-17 | 2000-02-16 | $7700.00 | Window Closed / Lapsed |