EP0185494A2
Expired / Lapsed
EP
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Detection of specific sequences in nucleic acids
Assignee: Applied Biosystems Inc
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Filing: 1985-12-06
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Granted: —
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Expires: 2005-12-06
Abstract
Abstract The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed. In the preferred mode, one of the probes is labeled so that the p ' esence or absence of the target sequence can then be tested by melting the sample nucleic acid-target probe duplex, eluting the dissociated target probe, and testing for the label. In another embodiment, the testing is accomplished without first removing probes not covalently attached, by attaching a hook to the probe that is not labeled, so that the labeled target probe may be recovered by catching the hook. In both instances, the presence of both the diagnostic probe and the contiguous probe is required for the label to appear in the assay. The above method is then applied to the detection of genetic diseases.
Inventors
- 👤 Norman M. Whiteley
- 👤 Michael W. Hunkapiller
- 👤 Alexander N. Glazer
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 17:21
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6862; C12Q1/6813; C12Q1/6816; C12Q1/6827; C12Q2600/00; C12Q2600/156; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S436/00; Y10S436/811
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
116 words
A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, which method comprises: a) treating the denatured nucleic acid with a probe complementary to a diagnostic portion of the target sequence, and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion, under conditions wherein the diagnostic probe remains bound substantially only to sample nucleic acid containing the target sequence; b) covalently attaching the diagnostic probe to the contiguous probe to give a target probe complementary to the target sequence; and c) removing the probes not attached in Step b) wherein at least one of the diagnostic and contiguous probes is labeled.
Claim 2
Independent Claim
116 words
A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, which method comprises: a) treating the denatured nucleic acid with a probe complementary to a diagnostic portion of the target sequence, and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion, under conditions wherein the diagnostic probe remains bound substantially only to sample nucleic acid containing the target sequence; b) covalently attaching the diagnostic probe to the contiguous probe to give a target probe complementary to the target sequence; and c) removing the probes not attached in Step b) wherein at least one of the diagnostic and contiguous probes is labeled.
Claim 3
Dependent Claim
10 words
The method of Claim 1 wherein the label is radioactive.
Claim 4
Dependent Claim
30 words
The method of Claim 2 wherein the label is 32 P at the 5 ' terminus of one probe to be ligated to the 3' terminus of the other probe.
Claim 5
Dependent Claim
24 words
The method of Claim 3 which further comprises: d) detecting the presence or absence of . radioactivity in the sample after conducting Step c).
Claim 6
Dependent Claim
26 words
The method of Claim 3 which further comprises: e) measuring the amount of radioactivity present in the sample after removing probes not attached in Step c).
Claim 7
Dependent Claim
12 words
The method of Claim 1 which further includes removing the target probe.
Claim 8
Dependent Claim
15 words
The method of Claim 6 wherein the target probe is eluted by increasing the stringency.
Claim 9
Dependent Claim
16 words
The method of Claim 7 wherein the target probe is eluted by using a temperature gradient.
Claim 10
Dependent Claim
20 words
The method of Claim 1 wherein a hook is attached to the unlabeled one of the diagnostic and contiguous probes.
Claim 11
Dependent Claim
15 words
The method of Claim 9 wherein the target probe is removed by catching the hook.
Claim 12
Dependent Claim
12 words
The method of Claim 10 wherein the hook comprises a biotin moiety.
Claim 13
Dependent Claim
18 words
The method of Claim 11 wherein the hook is caught by using streptavidin to bind the biotin moiety.
Claim 14
Independent Claim
152 words
A method of testing for the presence or absence of a target sequence in a sample containing DNA which method comprises: a) denaturing the sample DNA; b) treating the denatured DNA with a first probe which is complementary to a contiguous portion of the target sequence to give an aggregate of first probe and denatured DNA; c) treating the aggregate of b) with a second probe, which is shorter than the first probe, and which is complementary to a diagnostic portion of the target sequence; d) providing conditions of binding wherein the first probe does not bind substantially to the denatured DNA which does not contain the contiguous portion and wherein the second probe does not bind substantially to the denatured DNA which does not contain the diagnostic portion; e) ligating first probe and second probe; f) removing unligated probe; wherein at least one of the first and second probe is labeled.
Claim 15
Independent Claim
152 words
A method of testing for the presence or absence of a target sequence in a sample containing DNA which method comprises: a) denaturing the sample DNA; b) treating the denatured DNA with a first probe which is complementary to a contiguous portion of the target sequence to give an aggregate of first probe and denatured DNA; c) treating the aggregate of b) with a second probe, which is shorter than the first probe, and which is complementary to a diagnostic portion of the target sequence; d) providing conditions of binding wherein the first probe does not bind substantially to the denatured DNA which does not contain the contiguous portion and wherein the second probe does not bind substantially to the denatured DNA which does not contain the diagnostic portion; e) ligating first probe and second probe; f) removing unligated probe; wherein at least one of the first and second probe is labeled.
Claim 16
Independent Claim
108 words
A method for detecting a target sequence in a nucleic acid which method comprises: a) treating the denatured, immobilized nucleic acid sample with a first probe complementary to a contiguous portion of the target sequence under conditions of high stringency; b) treating the preparation of a) with a second- shorter probe complementary to a diagnostic portion of the target sequence under conditions of low stringency, wherein the second probe does not bind substantially to the denatured nucleic acid which does not contain the diagnostic portion; c) ligating first and second probe; and d) removing unligated probe. wherein at least one of the first and second probe .is labeled;
Claim 17
Independent Claim
108 words
A method for detecting a target sequence in a nucleic acid which method comprises: a) treating the denatured, immobilized nucleic acid sample with a first probe complementary to a contiguous portion of the target sequence under conditions of high stringency; b) treating the preparation of a) with a second- shorter probe complementary to a diagnostic portion of the target sequence under conditions of low stringency, wherein the second probe does not bind substantially to the denatured nucleic acid which does not contain the diagnostic portion; c) ligating first and second probe; and d) removing unligated probe. wherein at least one of the first and second probe .is labeled;
Claim 18
Dependent Claim
72 words
A diagnostic kit for detecting a target sequence in a nucleic acid sample, which kit comprises: a) a probe complementary to a diagnostic portion of the target sequence; b) a probe complementary to a nucleotide sequence contiguous with the diagnostic portion of the target sequence; wherein at least one of the probes of a) and b) is labeled; and wherein the probe of a) and the probe of b) can be ligated.
Claim 19
Dependent Claim
72 words
A diagnostic kit for detecting a target sequence in a nucleic acid sample, which kit comprises: a) a probe complementary to a diagnostic portion of the target sequence; b) a probe complementary to a nucleotide sequence contiguous with the diagnostic portion of the target sequence; wherein at least one of the probes of a) and b) is labeled; and wherein the probe of a) and the probe of b) can be ligated.
Claim 20
Dependent Claim
23 words
A kit as in claim 15 wherein the other of probes a) and b) which is not labeled has a hook attached thereto.
Claim 21
Dependent Claim
13 words
A kit as in Claim 16 further comprising means for catching the hook.
Claim 22
Independent Claim
216 words
A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid using a diagnostic probe which is complementary to a diagnostic portion of the target sequence and a contiguous probe which is complementary to a portion of said target sequence which is contiguous with said diagnostic portion, comprising the steps of: a) hybridizing the one of the diagnostic and contiguous probes which will remain bound to its complementary portion of the target sequence under the higher stringency, said one of the diagnostic and contiguous probes hereinafter called the HS probe; b) after Step a) hybridizing the other of said diagnostic and contiguous probes which will remain bound to its complementary portion in the target sequence under conditions of lower stringency, said other of said diagnostic and contiguous probes hereinafter called the LS probe; c) removing a substantial portion of said diagnostic probe which is not perfectly bound; d) covalently attaching said LS probe and said HS probe at sites on the nucleic acid where they are bound adjacent to each other to form a target probe complementary to said target sequence; e) removing labeled probe not attached in Step d); and f) testing for the presence of target probe in the sample remaining after Step e).
Claim 23
Independent Claim
216 words
A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid using a diagnostic probe which is complementary to a diagnostic portion of the target sequence and a contiguous probe which is complementary to a portion of said target sequence which is contiguous with said diagnostic portion, comprising the steps of: a) hybridizing the one of the diagnostic and contiguous probes which will remain bound to its complementary portion of the target sequence under the higher stringency, said one of the diagnostic and contiguous probes hereinafter called the HS probe; b) after Step a) hybridizing the other of said diagnostic and contiguous probes which will remain bound to its complementary portion in the target sequence under conditions of lower stringency, said other of said diagnostic and contiguous probes hereinafter called the LS probe; c) removing a substantial portion of said diagnostic probe which is not perfectly bound; d) covalently attaching said LS probe and said HS probe at sites on the nucleic acid where they are bound adjacent to each other to form a target probe complementary to said target sequence; e) removing labeled probe not attached in Step d); and f) testing for the presence of target probe in the sample remaining after Step e).
Claim 24
Dependent Claim
18 words
A method as in Claim 18 further comprising the step of: g) measuring the amount of target probe.
Claim 25
Dependent Claim
24 words
A method as in Claim 19 wherein one of said diagnostic and contiguous probes contains a label and Step f) includes detecting said label.
Claim 26
Dependent Claim
18 words
A method as in Claim 20 wherein Step f) comprises increasing the stringency to remove the target probe.
Claim 27
Dependent Claim
22 words
A method as in Claim 21 wherein Step f) further comprises measuring the amount of label removed in removing the target probe.
Claim 28
Dependent Claim
27 words
A method as in Claim 18 wherein Step d) comprises covalently attaching the 5' end of said diagnostic probe with the 3' end of said contiguous probe.
Claim 29
Dependent Claim
13 words
A method as in Claim 23 wherein Step d) is conducted using ligase.
Claim 30
Dependent Claim
27 words
A method as in Claim 18 wherein Step d) comprises covalently attaching the 5' end of the contiguous probe with the 3' end of the diagnostic probe.
Claim 31
Dependent Claim
13 words
A method as in Claim 25 wherein Step d) is conducted using ligase.
Claim 32
Dependent Claim
49 words
A method as in Claim 16 wherein said target sequence corresponds to a genetic mutation from a known normal sequence, said method further comprising the step of comparing said amount of target probe from Step g) with a calibrated standard for nucleic acid known to have said genetic mutation.
References Cited (Backward Citations)
No citation records indexed.
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