US4775619A
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US
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Polynucleotide determination with selectable cleavage sites
Assignee: Bayer Corp
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Filing: 1984-10-16
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Granted: 1988-10-04
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Expires: 2004-10-16
Abstract
Abstract Methods for diagnosis employing polynucleotides having oligonucleotide sequences substantially homologous to a sequence of interest, where the presence or absence of hybridization at a predetermined stringency provides for the release of a label from a support. Particularly, various techniques are employed for binding a label to a support, whereupon cleavage of either a single or double strand, a label may be released from a support, where the release of the label can be detected as indicative of the presence of a particular oligonucleotide sequence in a sample. The method finds use in diagnosis of disease, genetic monitoring, and analyzing nucleic acid mixtures.
Inventors
- 👤 Mickey S. Urdea
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 04:07
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/6816; C12Q1/6823; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/81
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
135 words
A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising: combining under hybridizing conditions said nucleic acid sample with a polynucelotide reagent, wherein one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynuceleotide reagent results in a label being bound to said support through a selectable cleavage site, wherein said selectable cleavage site is a restriction site resulting from the formation of a duplex at the oligonucleotide sequence of interest; substantially freeing said support of label bound to said support other than through said selectable cleavage site; cleaving said cleavage site with a restriction endonuclease which recognizes said site; and detecting label free of said support.
Claim 2
Independent Claim
135 words
A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising: combining under hybridizing conditions said nucleic acid sample with a polynucelotide reagent, wherein one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynuceleotide reagent results in a label being bound to said support through a selectable cleavage site, wherein said selectable cleavage site is a restriction site resulting from the formation of a duplex at the oligonucleotide sequence of interest; substantially freeing said support of label bound to said support other than through said selectable cleavage site; cleaving said cleavage site with a restriction endonuclease which recognizes said site; and detecting label free of said support.
Claim 3
Dependent Claim
21 words
A method according to claim 1, wherein said hybridization is effected through the binding of at least 4 complementary base pairs.
Claim 4
Dependent Claim
18 words
A method according to claim 1, wherein said polynucelotide reagent comprises a labeled polynucleotide bound to a support.
Claim 5
Dependent Claim
113 words
A method according to claim 1, wherein said polynucleotide reagent comprises a first polynucleotide bound to a support and a labeled second polynucleotide, wherein said first and second polynucleotides have oligonucleotide sequences complementary to sequences present in said analyte to form duplexes under said hybridizing conditions, at least one of said oligonucleotide sequence being a sequence of interest, wherein (1) either of said duplexes defines a restriction site, as said selectable cleavage site and said cleavage is with a restriction endonulease which recognizes said site, or (2) said label is linked to said polynucleotide through a selectable cleavage site and said cleavage is by cleaving said selectable cleavage site enzymatically or with light.
Claim 6
Dependent Claim
23 words
A method according to claim 1, wherein said nucleic acid sample is bound to said support and said polynucleotide reagent is labeled polynucleotide.
Claim 7
Dependent Claim
12 words
A method according to claim 1, wherein said label is a radionuclide.
Claim 8
Dependent Claim
12 words
A method according to claim 1, wherein said label is a fluorescer.
Claim 9
Dependent Claim
12 words
A method according to claim 1, wherein said label is an enzyme.
Claim 10
Dependent Claim
15 words
A method according to claim 1, wherein said label is an enzyme substrate or cofactor.
Claim 11
Dependent Claim
32 words
A method according to claim 1, wherein there is more than one label bound to said support through a plurality of selectable cleavage sites; and a plurality of cleavage sites are cleaved.
Claim 12
Dependent Claim
21 words
A method according to claim 1, wherein said hybridization is effected through the binding of at least 12 complementary base pairs.
Claim 13
Independent Claim
165 words
A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising: combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with a polynucleotide reagent, where one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through a selectable cleavage site, wherein said selectable cleavage site is a restriction site resulting from the formation of a duplex at the oligonucleotide sequence of interest; separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium; washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said selectable cleavage site; cleaving said cleavage site with a restriction endonuclease which recognizes said site; and detecting label free of said support.
Claim 14
Independent Claim
165 words
A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising: combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with a polynucleotide reagent, where one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through a selectable cleavage site, wherein said selectable cleavage site is a restriction site resulting from the formation of a duplex at the oligonucleotide sequence of interest; separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium; washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said selectable cleavage site; cleaving said cleavage site with a restriction endonuclease which recognizes said site; and detecting label free of said support.
Claim 15
Dependent Claim
18 words
A method according to claim 11, wherein said polynucleotide reagent comprises a labelled polynucleotide bound to a support.
Claim 16
Dependent Claim
95 words
A method according to claim 11, wherein said polynucleotide reagent comprises a first polynucleotide bound to a support and a labeled second polynucleotide, wherein said first and second polynucleotides have oligonucleotide sequences complementary to sequences present in said analyte to form duplexes, at least one of said oligonucleotide sequences being a sequence of interest, wherein (1) either of said duplexes defines a restriction site as said selectable cleavage site and said cleavage is with a restriction endonuclease which recognizes said site, or (2) said label is linked to said polynucleotide through a selectable cleavage site.
Claim 17
Dependent Claim
23 words
A method according to claim 14, wherein said nucleic acid sample is bound to said support and said polynucleotide reagent is labeled polynucleotide.
Claim 18
Dependent Claim
21 words
A method according to claim 12, wherein said hybridization is effected through the binding of at least 4 complementary base pairs.
Claim 19
Dependent Claim
21 words
A method according to claim 12, wherein said hybridization is effected through the binding of at least 12 complementary base pairs.
Claim 20
Dependent Claim
81 words
A reagent for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a polynucleotide sequence bound proximal to one end to a support and proximal to the opposite end to a label capable of providing, directly or indirectly, a detectable signal, said polynucleotide sequence having a sequence which when duplexed with said sequence of interest defines a selectable cleavage site cleavable by a restriction endonuclease which recognizes said site.
Claim 21
Dependent Claim
81 words
A reagent for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a polynucleotide sequence bound proximal to one end to a support and proximal to the opposite end to a label capable of providing, directly or indirectly, a detectable signal, said polynucleotide sequence having a sequence which when duplexed with said sequence of interest defines a selectable cleavage site cleavable by a restriction endonuclease which recognizes said site.
Claim 22
Dependent Claim
146 words
A reagent composition for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a first polynucleotide having a first oligonucleotide sequence complementary to a first portion of said sequence of interest; and a labeled second polynucleotide, wherein said label provides, directly or indirectly a detectable signal, said second labeled polynucleotide having a second oligonucleotide sequence complementary to a second portion of said sequence of interest, said first and second portions comprising the entirety of said sequence of interest, wherein a continuous duplex may be formed, under hybridizing conditions, between said first oligonucleotide sequence and said first portion of said sequence of interest and between said second oligonucleotide sequence and said second portion of said sequence of interest, said continuous duplex containing a selectable cleavage site cleavage by a restriction endonuclease which recognizes said site.
Claim 23
Dependent Claim
146 words
A reagent composition for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a first polynucleotide having a first oligonucleotide sequence complementary to a first portion of said sequence of interest; and a labeled second polynucleotide, wherein said label provides, directly or indirectly a detectable signal, said second labeled polynucleotide having a second oligonucleotide sequence complementary to a second portion of said sequence of interest, said first and second portions comprising the entirety of said sequence of interest, wherein a continuous duplex may be formed, under hybridizing conditions, between said first oligonucleotide sequence and said first portion of said sequence of interest and between said second oligonucleotide sequence and said second portion of said sequence of interest, said continuous duplex containing a selectable cleavage site cleavage by a restriction endonuclease which recognizes said site.
Claim 24
Dependent Claim
76 words
A deoxyribonucleotide reagent comprising a deoxyribonucleotide sequence of at least four bases joined to a glass particle support through the reaction product of a semicarbazide and the dialdehyde reaction product of a ribonuceleotide and an oxidant, wherein said at least four bases are selected so as to form a duplex with a sequence of interest under hybridizing conditions, the duplex so formed containing a selectable cleavage site cleavable with a restriction endonuclease which recognizes said site.
Claim 25
Dependent Claim
76 words
A deoxyribonucleotide reagent comprising a deoxyribonucleotide sequence of at least four bases joined to a glass particle support through the reaction product of a semicarbazide and the dialdehyde reaction product of a ribonuceleotide and an oxidant, wherein said at least four bases are selected so as to form a duplex with a sequence of interest under hybridizing conditions, the duplex so formed containing a selectable cleavage site cleavable with a restriction endonuclease which recognizes said site.
Claim 26
Dependent Claim
82 words
A deoxyribonucleotide reagent comprising a deoxyribonucleotide sequence of at least four bases joined to a glass support through a phosphothiacetamide link, said link formed by reaction between a bromoacetamide substitutent on said glass support and a 5'-thophosphate moiety on said deoxyribonucleotide reagent, wherein said at least four bases are selected so as to form a duplex with a sequence of interest under hybridizing conditions, the duplex so formed containing a selectable cleavage site cleavable with a restriction endonuclease which recognizes said site.
Claim 27
Dependent Claim
82 words
A deoxyribonucleotide reagent comprising a deoxyribonucleotide sequence of at least four bases joined to a glass support through a phosphothiacetamide link, said link formed by reaction between a bromoacetamide substitutent on said glass support and a 5'-thophosphate moiety on said deoxyribonucleotide reagent, wherein said at least four bases are selected so as to form a duplex with a sequence of interest under hybridizing conditions, the duplex so formed containing a selectable cleavage site cleavable with a restriction endonuclease which recognizes said site.
Claim 28
Dependent Claim
18 words
The reagent composition of claim 21, wherein said duplex is formed from at least 12 complementary base pairs.
Claim 29
Dependent Claim
115 words
A reagent composition for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a first component which is a polynucleotide bound to a support and a second component which is a labeled second polynucleotide, wherein said label provides, directly or indirectly, a detectable signal, wherein said first and said second polynucleotides have oligonucleotide sequences complementary to sequences present in said analyte which form duplexes with said analyte under hybridizing conditions, at least one of said oligonucleotide sequences being complementary to a sequence of interest, wherein one of said duplexes defines a selectable cleavage site cleavable by a restriction endonuclease which recognizes said site.
Claim 30
Dependent Claim
115 words
A reagent composition for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a first component which is a polynucleotide bound to a support and a second component which is a labeled second polynucleotide, wherein said label provides, directly or indirectly, a detectable signal, wherein said first and said second polynucleotides have oligonucleotide sequences complementary to sequences present in said analyte which form duplexes with said analyte under hybridizing conditions, at least one of said oligonucleotide sequences being complementary to a sequence of interest, wherein one of said duplexes defines a selectable cleavage site cleavable by a restriction endonuclease which recognizes said site.
Claim 31
Dependent Claim
18 words
The reagent composition of claim 23, wherein said duplexes are formed from at least 4 complementary base pairs.
Claim 32
Dependent Claim
18 words
The reagent composition of claim 23, wherein said duplexes are formed from at least 12 complementary base pairs.
References Cited (Backward Citations)
US4032659A
Cited
US4146454A
Cited
US4230797A
Cited
US4419444A
Cited
US4469796A
Cited
US4358535A
Cited
US4358535B1
Cited
US4430432A
Cited
EP0070687B1
Cited
US4395486A
Cited
US4486539A
Cited
US4563419A
Cited
US4542102A
Cited
US4683194A
Cited
US4563417A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-10-04 | 1992-04-04 | 1992-10-04 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-10-04 | 1996-04-04 | 1996-10-04 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-10-04 | 2000-04-04 | 2000-10-04 | $7700.00 | Window Closed / Lapsed |