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Patent US4656127A successfully refreshed from Google Patents.
US4656127A
Expired / Lapsed
US
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Method of detecting mutations in DNA and RNA
Assignee: GE Healthcare Ltd
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Filing: 1984-04-23
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Granted: 1987-04-07
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Expires: 2004-04-23
Abstract
Abstract A method of detecting a mutation of a specific nucleotide base in a target nucleic acid chain comprises: (a) hybridizing a labelled probe to the target to form a hybrid in which one end of the probe is positioned adjacent the specific base; (b) adding a nucleotide derivative, e.g. a thionucleotide, under conditions to cause it to join to the end of the probe if it is complementary to the specific base; (c) digesting the hybrid using an exonuclease enzyme under conditions such that the nucleotide derivative protects the probe from digestion; and observing the presence or absence of label attached to the target. The method can be used to detect mutations even when these are not present at restriction enzyme cleavage sites, and does not require the preliminary steps of restriction digestion, gel electrophoresis and DNA (or RNA) blotting.
Inventors
- 👤 Christopher R. Mundy
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 18:41
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6858; C12N; C12N15/00
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
223 words
A method of detecting a mutation of a specific nucleotide base in a target nucleic acid chain by providing a linear probe complementary to a part of the nucleic acid chain extending in one direction from the specific base, (a) hybridizing the probe to the target to form a nucleic acid hybrid, whereby one end of the probe becomes hybridized to the nucleic acid chain substantially adjacent the specific base, (b) admixing with the hybrid a nucleotide derivative, under conditions appropriate for probe extension, so as to cause the nucleotide derivative to join on to the end of the probe only if the specific base in the target is, or is not, the mutation to be detected, a probe carrying said nucleotide derivative being resistant to digestion under particular conditions, wherein one of the probe and the nucleotide derivative is labelled, (c) subjecting the hybrid to digestion by an exonuclease enzyme under the said conditions whereby the double-stranded portion thereof is progressively digested starting at the said end of the probe unless the end has had said nucleotide derivative joined to it, (d) removing portions of the probe which are no longer hybridized to the nucleic acid chain, (e) and using the presence or absence of the probe remaining after digestion to detect a mutation of the specific nucleotide base in the target.
Claim 2
Independent Claim
223 words
A method of detecting a mutation of a specific nucleotide base in a target nucleic acid chain by providing a linear probe complementary to a part of the nucleic acid chain extending in one direction from the specific base, (a) hybridizing the probe to the target to form a nucleic acid hybrid, whereby one end of the probe becomes hybridized to the nucleic acid chain substantially adjacent the specific base, (b) admixing with the hybrid a nucleotide derivative, under conditions appropriate for probe extension, so as to cause the nucleotide derivative to join on to the end of the probe only if the specific base in the target is, or is not, the mutation to be detected, a probe carrying said nucleotide derivative being resistant to digestion under particular conditions, wherein one of the probe and the nucleotide derivative is labelled, (c) subjecting the hybrid to digestion by an exonuclease enzyme under the said conditions whereby the double-stranded portion thereof is progressively digested starting at the said end of the probe unless the end has had said nucleotide derivative joined to it, (d) removing portions of the probe which are no longer hybridized to the nucleic acid chain, (e) and using the presence or absence of the probe remaining after digestion to detect a mutation of the specific nucleotide base in the target.
Claim 3
Dependent Claim
31 words
The method as claimed in claim 1, wherein the probe is provided such that in step (a) one end becomes hybridized to the nucleic acid chain immediately adjacent the specific base.
Claim 4
Dependent Claim
62 words
The method as claimed in claim 2, wherein step (b) is: (b) admixing with the hybrid a nucleotide derivative under conditions appropriate for probe extension so as to cause the nucleotide derivative to join on to the end of the probe if it is complementary to the specific base, a probe carrying said nucleotide derivative being resistant to digestion under particular conditions.
Claim 5
Dependent Claim
104 words
The method as claimed in claim 2, wherein step (b) is: (b)(i) admixing with the hybrid a chain-terminating nucleotide compound under conditions appropriate for probe extension so as to cause it to join on to the end of the probe if it is complementary to the specific base, (b)(ii) admixing with the resulting hybrid one or more nucleotide derivatives under conditions appropriate for probe extension so as to cause them to join on to the end of the probe if a chain-terminating nucleotide compound is not already present, a probe carrying said one or more nucleotide derivatives being resistant to digestion under particular conditions.
Claim 6
Dependent Claim
34 words
The method as claimed in claim 1, wherein the probe is provided such that in step (a) one end becomes hybridized to the nucleic acid chain a few bases away from the specific base.
Claim 7
Dependent Claim
71 words
The method as claimed in claim 5, wherein step (b) is: (b) admixing the hybrid a nucleotide derivative, together with one or two other different nucleotides, under conditions appropriate for probe extension so as to cause them to join on to the end of the probe, including the nucleotide derivative if it is complementary to the specific base, a probe carrying said nucleotide derivative being resistant to digestion under particular conditions.
Claim 8
Dependent Claim
117 words
The method as claimed in claim 5, wherein step (b) is: (b)(i) admixing with the hybrid a chain-terminating nucleotide compound, together with one or two other different nucleotides, under conditions appropriate for probe extension so as to cause them to join on to the end of the probe, including the chain-terminating nucleotide compound if it is complementary to the specific base, (b)(ii) admixing with the resulting hybrid one or more nucleotide derivatives under conditions appropriate for probe extension so as to cause them to join on to the end of the probe if a chain-terminating nucleotide compound is not already present, a probe carrying said one or more nucleotide derivatives being resistant to digestion under particular conditions.
Claim 9
Dependent Claim
14 words
The method as claimed in claim 1, wherein the target is DNA or RNA.
Claim 10
Dependent Claim
12 words
The method as claimed in claim 1, wherein the probe is labelled.
Claim 11
Dependent Claim
14 words
The method as claimed in claim 1, wherein the probe is DNA or RNA.
Claim 12
Dependent Claim
18 words
The method as claimed in claim 1, wherein the nucleotide derivative used in step (b) is a thionucleotide.
Claim 13
Dependent Claim
28 words
A method as claimed in claim 1, wherein there is used in step (c) an exonuclease enzyme that digests double-stranded nucleic acid chains only from the 3' ends.
Claim 14
Dependent Claim
24 words
The method as claimed in claim 1, wherein the probe extension is achieved by the addition of calf thymus DNA polymerase under polymerisation conditions.
Claim 15
Dependent Claim
12 words
A method as claimed in claim 1, wherein the target is immobilised.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-04-06 | 1990-10-06 | 1991-04-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-04-06 | 1994-10-06 | 1995-04-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-04-06 | 1998-10-06 | 1999-04-07 | $7700.00 | Window Closed / Lapsed |