Patent EP0237362B1 successfully refreshed from Google Patents.
EP0237362B1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids and kits therefor
Assignee: F Hoffmann La Roche AG
•
Filing: 1987-03-13
•
Granted: 1992-03-11
•
Expires: 2007-03-13
Abstract
Abstract A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids in a sample comprises: (a) treating the sample, together or sequentially with 4 different nucleotide triphosphates (I), an agent (II) for polymerisation of (I), and one oligonucleotide primer for each strand of each nucleic acid suspected of contg. the variation(s) under hybridising conditions, such that for each strand contg. each different variation, an extension prod. of each primer is sinthesised which is complementary to each strand, where the primer(s) are selected to be complementary to each strand contg. each variation, such that the extension prod. synthesised from one primer, when sepd. from its complement, can serve as a template for synthesis of the extension prod. of the other primer; (b) treating the sample under denaturing conditions to separate primer extension prods. from their templates if the variation(s) are present (c) treating the sample, together or sequentially, with the four (I), (II) and oligonucleotide primers such that primer extension prod. is synthesised using each of the single strands produced in (b), and repeating (b) and (c) to give detectable amplification of the nucleic acid contg. the variation(s), if present; (d) affixing the prod. of (c) to a membrane; (e) treating the membrane under hybridisation conditions with a labelled sequence -specific oligonucleotide probe capable of hybridising with the amplified nucleic acid contg. the variation(s), if present; (d) affixing the prod. of (c) to a membrane; (e) treating the membrane under hybridisation conditions with a labelled sequence-specific oligonucleotide probe capable of hybridising with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and (f) detecting the presence of hybridisation.
Inventors
- 👤 Henry Anthony Erlich
- 👤 Randall Keichi Saiki
- 👤 Glenn Thomas Horn
- 👤 Kary Banks Mullis
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 12:44
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6834; C12Q1/6837; C07; C07K; C07K14/00; C07K14/435; C07K14/705; C07K14/70503; C07K14/70539; C12Q1/6827; C12Q1/6844; C12Q1/6858; C12Q1/686; C12Q1/6876; C12Q1/6881; C12Q1/6883; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
269 words
A process for detecting the presence of a specific nucleotide sequence in nucleic acid in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and two oligonucleotide primers for said nucleic acid under hybridizing conditions such that a primer will hybridize to said nucleic acid and an extension product of the primer be synthesized which is complementary to said nucleic acid, wherein said primers are selected such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates; (c) treating the sample, together or sequentially, with said four nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, wherein steps (b) and (c) are repeated a sufficient number of times exponentially to increase the amount of said nucleic acid and to result in detectable amplification thereof: (d) directly transferring, without gel fractionation, product derived from step (c) to a membrane; (e) treating the membrane from (d) under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid; and (f) detecting whether the probe has hybridized to an amplified nucleic acid in the sample.
Claim 2
Dependent Claim
269 words
A process for detecting the presence of a specific nucleotide sequence in nucleic acid in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and two oligonucleotide primers for said nucleic acid under hybridizing conditions such that a primer will hybridize to said nucleic acid and an extension product of the primer be synthesized which is complementary to said nucleic acid, wherein said primers are selected such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates; (c) treating the sample, together or sequentially, with said four nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, wherein steps (b) and (c) are repeated a sufficient number of times exponentially to increase the amount of said nucleic acid and to result in detectable amplification thereof: (d) directly transferring, without gel fractionation, product derived from step (c) to a membrane; (e) treating the membrane from (d) under hybridization conditions with a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid; and (f) detecting whether the probe has hybridized to an amplified nucleic acid in the sample.
Claim 3
Dependent Claim
49 words
A process of Claim 1, wherein the nucleic acid in the sample is extracted from the sample prior to step (a), and steps (b) and (c) are repeated at least five times, optionally wherein the sample contains human genomic DNA and steps (b) and (c) are repeated 15-30 times.
Claim 4
Dependent Claim
26 words
A process of Claim 1 or Claim 2, wherein said nucleic acid is double-stranded and its strands are separated by denaturing before or during step (a).
Claim 5
Dependent Claim
15 words
A process of Claim 1 or Claim 2, wherein the said nucleic acid is single-stranded.
Claim 6
Dependent Claim
16 words
A process of Claim 3, wherein said nucleic acid is DNA and said primers are oligodeoxyribonucleotides.
Claim 7
Dependent Claim
19 words
A process of Claim 4, wherein said nucleic acid is DNA or messenger RNA and said primers are oiigodeoxyribonudeotides.
Claim 8
Dependent Claim
40 words
A process of any one of Claims 1 to 6, wherein said agent for polymerization is selected from E coli DNA Polymerase I, Klenow fragment of E coli DNA Polymerase I, T4 DNA polymerase, thermostable polymerase enzyme and reverse transcriptase.
Claim 9
Dependent Claim
75 words
A process of any one of Claims 1 to 7, wherein the specific nucleotide sequence is contained within a human nucleic acid selected from a beta-globin gene, an oncogene, an HLA-DQ alpha, DQ beta, DR beta, DP alpha and DP beta gene, and mitochondrial DNA, optionally wherein the nucleotide sequence is associated with insulin-dependent diabetes mellitus or wherein the nucleotide sequence is contained within nucleic acid of an organism that can cause an infectious disease.
Claim 10
Dependent Claim
71 words
A process of any one of Claims 1 to 8, wherein either the sample comprises cells, optionally wherein the sample is peripheral blood lymphocytes or amniotic fluid, and the process further comprises, before step (a), the step of heating the sample sufficiently to expose the nucleic acid(s) therein; or wherein the sample is a semen, hair or blood sample, or contains mitochondrial DNA, and the process is used in forensic analysis.
Claim 11
Dependent Claim
285 words
A process for detecting the presence of a specific nucleotide sequence in a nucleic acid in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and two oligonucleotide primers for said nucleic acid under hybridizing conditions such that a primer hybridizes to said nucleic acid and an extension product of the primer is synthesized which is complementary to said nucleic acid, wherein said primers are selected such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates; (c) treating the sample, together or sequentially, with said four nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, wherein steps (b) and (c) are repeated a sufficient number of times exponentially to increase the amount of said nucleic acid and to result in detectable amplification thereof; and wherein at least one primer and/or at least one of the four nucleoside triphosphates is labeled with a detectable moiety; (d) treating a membrane to which is attached a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid only if a sequence of the oligonucleotide is complementary to a region of the amplified nucleic acid under hybridization conditions with product derived from step (c); and (e) detecting whether an amplified nucleic acid in the sample has hybridized to the oligonucleotide attached to the membrane.
Claim 12
Dependent Claim
285 words
A process for detecting the presence of a specific nucleotide sequence in a nucleic acid in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and two oligonucleotide primers for said nucleic acid under hybridizing conditions such that a primer hybridizes to said nucleic acid and an extension product of the primer is synthesized which is complementary to said nucleic acid, wherein said primers are selected such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates; (c) treating the sample, together or sequentially, with said four nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, wherein steps (b) and (c) are repeated a sufficient number of times exponentially to increase the amount of said nucleic acid and to result in detectable amplification thereof; and wherein at least one primer and/or at least one of the four nucleoside triphosphates is labeled with a detectable moiety; (d) treating a membrane to which is attached a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid only if a sequence of the oligonucleotide is complementary to a region of the amplified nucleic acid under hybridization conditions with product derived from step (c); and (e) detecting whether an amplified nucleic acid in the sample has hybridized to the oligonucleotide attached to the membrane.
Claim 13
Dependent Claim
306 words
A process for detecting the presence of a specific nucleotide sequence in nucleic acid in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and two oligonucleotide primers for said nucleic acid under hybridizing conditions such that a primer hybridizes to said nucleic acid and an extension product of the primer is synthesized which is complementary to said nucleic acid, wherein said primers are selected such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates; (c) treating the sample, together or sequentially, with said four nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, wherein steps (b) and (c) are repeated a sufficient number of times exponentially to increase the amount of said nucleic acid and to result in detectable amplification thereof; (d) attaching to a membrane a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid; (e) treating the membrane from (d) under hybridization conditions with product derived from step (c): (f) treating the product of step (e) with a restriction enzyme which will cleave any hybrids formed if the probe and any sequence to be detected both contain a restriction site recognized by the enzyme; and (g) detecting whether a labeled restriction fragment of the required length is present in the restriction digest thereby indicating that hybridization occurred.
Claim 14
Dependent Claim
306 words
A process for detecting the presence of a specific nucleotide sequence in nucleic acid in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and two oligonucleotide primers for said nucleic acid under hybridizing conditions such that a primer hybridizes to said nucleic acid and an extension product of the primer is synthesized which is complementary to said nucleic acid, wherein said primers are selected such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) treating the sample under denaturing conditions to separate the primer extension products from their templates; (c) treating the sample, together or sequentially, with said four nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, wherein steps (b) and (c) are repeated a sufficient number of times exponentially to increase the amount of said nucleic acid and to result in detectable amplification thereof; (d) attaching to a membrane a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid; (e) treating the membrane from (d) under hybridization conditions with product derived from step (c): (f) treating the product of step (e) with a restriction enzyme which will cleave any hybrids formed if the probe and any sequence to be detected both contain a restriction site recognized by the enzyme; and (g) detecting whether a labeled restriction fragment of the required length is present in the restriction digest thereby indicating that hybridization occurred.
Claim 15
Dependent Claim
23 words
A process of Claim 10, wherein said primers are oligodeoxyribonucleotides and are optionally labeled and said agent for polymerization is a DNA polymerase.
Claim 16
Dependent Claim
18 words
A process of Claim 10 or Claim 12, wherein said nucleic acid is DNA, optionally human genomic DNA.
Claim 17
Dependent Claim
14 words
A process of Claim 10 or Claim 12, wherein said nucleic acid is RNA.
Claim 18
Dependent Claim
43 words
A process of Claim 13, wherein said primers amplify a segment of a Class II HLA gene, optionally a DQ alpha allele, a DQ beta allele, a DP alpha allele, a DP beta allele, a DR alpha allele, or a DR beta allele.
Claim 19
Dependent Claim
178 words
A kit for detecting the presence of a specific nucleotide sequence in a nucleic acid in a sample, which kit comprises, in packaged form, a multicontainer unit having: (a) two oligonucleotide primers for said nucleic acid, which primers are selected such that an extension product synthesized from one primer, when separated from its complement, can serve as a template for the synthesis of the extension product of the other primer so as exponentially to amplify said nucleic acid; (b) an agent for polymerization; (c) four different nucleoside triphosphates; and either: (d) a sequence specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid; and (e) a membrane to which amplified nucleic acid can bind for subsequent detection by probe hybridization; or (d) a sequence specific oligonucleotide probe attached to a membrane, wherein said probe is capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid.
Claim 20
Dependent Claim
178 words
A kit for detecting the presence of a specific nucleotide sequence in a nucleic acid in a sample, which kit comprises, in packaged form, a multicontainer unit having: (a) two oligonucleotide primers for said nucleic acid, which primers are selected such that an extension product synthesized from one primer, when separated from its complement, can serve as a template for the synthesis of the extension product of the other primer so as exponentially to amplify said nucleic acid; (b) an agent for polymerization; (c) four different nucleoside triphosphates; and either: (d) a sequence specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid; and (e) a membrane to which amplified nucleic acid can bind for subsequent detection by probe hybridization; or (d) a sequence specific oligonucleotide probe attached to a membrane, wherein said probe is capable of hybridizing with the amplified nucleic acid only if a sequence of the probe is complementary to a region of the amplified nucleic acid.
Claim 21
Dependent Claim
43 words
A kit of Claim 16, wherein either said primers are oligodeoxyribonucleotides, said nucleoside triphosphates are dATP, dCTP, dGTP and TTP, and the probe is labeled or wherein at least one of the primers and/or at least one of the nucleoside triphosphates is labeled.
Claim 22
Dependent Claim
66 words
A kit of Claim 16 or Claim 17, further comprising means of separating the strands of any double-stranded nucleic acids contained in the sample, and/or a positive control for the probe which contains one or more nucleic acids with the specific nucleotide sequence to be detected and a negative control for the probe which does not contain nucleic acid with the nucleotide sequence to be detected.
Claim 23
Dependent Claim
44 words
A kit of any one of Claims 16 to 18, wherein the agent for polymerization is an enzyme selected from E coli DNA Polymerase I, Klenow fragment of E coli DNA Polymerase I, T4 DNA polymerase, a heat-stable DNA polymerase enzyme, and reverse transcriptase.
Claim 24
Dependent Claim
55 words
A kit of any one of Claims 16 to 19 wherein the specific nucleotide sequence is contained within a human nucleic acid selected from a beta-globin gene, an HLA-DQ alpha, DQ beta, DR beta, DP alpha and DP beta gene, and mitochondrial DNA, optionally the nucleotide variation in sequence being associated with insulin-dependent diabetes mellitus.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.