Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids and kits therefor

Claim 2

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleotide triphosphates, an agent for polymerization of the nucleotide triphosphates, and one oligonucleotide primer for each strand of each nucleic acid suspected of containing said variation(s) under hybridizing conditions, such that for each nucleic acid strand containing each different variation to be detected, an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each nucleic acid strand containing each different variation, such that the extension product synthesized from one primer, when it is 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 if the variation(s) to be detected are present; (c) treating the sample, together or sequentially, with said four nucleotide triphosphates, an agent for polymerization of the nucleotide 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 to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present; (d) affixing the product of step (c) to a membrane; (e) treating the membrane under hybridization conditions With a labeled sequence-specific oligonucleotide probe capable of hybridizing 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 whether the probe has hybridized to an amplified sequence in the nucleic acid sample.

Claim 3

A process according to claim 1, wherein -the nucleic acid(s) contained in the sample are extracted from the sample prior to step (a) and steps (b) and (c) are carried out at least five times.

Claim 4

A process according to claim 1 or 2, wherein steps (a) and (c) are accomplished using an enzyme selected from the group consisting of E. coli DNA Polymerase I, Klenow fragment of E. coli DNA Polymerase I, T4 DNA polymerase, thermostable enzyme, and reverse transcriptase.

Claim 5

A process according to any one of claims 1-3, wherein the nucleotide variation in sequence is associated with a cancerous or infectious disease, sickle cell anemia, hemoglobin C disease, the human HLA-DQa, DQS, DRS, DPα or DPβ region, or mitochondrial DNA.

Claim 6

A process according to any one of claims 1-4, wherein the sample comprises cells, and the process further comprises, before step (a), the step of heating the sample sufficiently to expose the nucleic acid(s) therein.

Claim 7

A process according to any one of claims 1-5, wherein the sample is a semen, hair or blood sample, or contains mitochondrial DNA, and the process is used in forensic analysis.

Claim 8

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleotide triphosphates, an agent for polymerization of the nucleotide triphosphates, and one oligonucleotide primer for each strand of each nucleic acid suspected of containing said variation under hybridizing conditions, such that for each nucleic acid strand containing each different variation to be detected, an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each nucleic acid strand containing each different variation, such that the extension product synthesized from one primer, when it is 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 if the variation(s) to be detected are present; (c) treating the sample, together or sequentially, with said four nucleotide triphosphates, an agent for polymerization of the nucleotide 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 to result in detectable amplification of the nucleic acid containing the sequence (variation(s), if present, and wherein at least one primer and/or at least one of the four nucleotide triphosphates is labeled with a detectable moiety; (d) affixing to a membrane a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the oligonucleotide is complementary to a region of the amplified sequence; (e) treating the membrane under hybridization conditions with the product of step (c); and (f) detecting whether an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane.

Claim 9

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleotide triphosphates, an agent for polymerization of the nucleotide triphosphates, and one oligonucleotide primer for each strand of each nucleic acid suspected of containing said variation under hybridizing conditions, such that for each nucleic acid strand containing each different variation to be detected, an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each nucleic acid strand containing each different variation, such that the extension product synthesized from one primer, when it is 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 if the variation(s) to be detected are present; (c) treating the sample, together or sequentially, with said four nucleotide triphosphates, an agent for polymerization of the nucleotide 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 to result in detectable amplification of the nucleic acid containing the sequence (variation(s), if present, and wherein at least one primer and/or at least one of the four nucleotide triphosphates is labeled with a detectable moiety; (d) affixing to a membrane a sequence-specific oligonucleotide capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the oligonucleotide is complementary to a region of the amplified sequence; (e) treating the membrane under hybridization conditions with the product of step (c); and (f) detecting whether an amplified sequence in the nucleic acid sample has hybridized to the oligonucleotide affixed to the membrane.

Claim 10

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleotide triphosphates, an agent for polymerization of the nucleotide triphosphates, and one oligonucleotide primer for each strand of each nucleic acid suspected of containing said variation under hybridizing conditions, such that for each nucleic acid strand containing each different variation to be detected, an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each nucleic acid strand containing each different variation, such that the extension product synthesized from one primer, when it is 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 if the variation(s) to be detected are present; (c) treating the sample, together or sequentially, with said four nucleotide triphosphates, an agent for polymerization of the nucleotide 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 to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present; (d) affixing to a membrane a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; (e) treating the membrane under hybridization conditions with the product of step (c); (f) treating the product of step (e) with a restriction enzyme which will cleave any hybrids formed if the probe and variation(s) 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 to indicate that hybridization occurred.

Claim 11

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, which process comprises: (a) treating the sample, together or sequentially, with four different nucleotide triphosphates, an agent for polymerization of the nucleotide triphosphates, and one oligonucleotide primer for each strand of each nucleic acid suspected of containing said variation under hybridizing conditions, such that for each nucleic acid strand containing each different variation to be detected, an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primer or primers are selected so as to be substantially complementary to each nucleic acid strand containing each different variation, such that the extension product synthesized from one primer, when it is 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 if the variation(s) to be detected are present; (c) treating the sample, together or sequentially, with said four nucleotide triphosphates, an agent for polymerization of the nucleotide 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 to result in detectable amplification of the nucleic acid containing the sequence variation(s), if present; (d) affixing to a membrane a labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; (e) treating the membrane under hybridization conditions with the product of step (c); (f) treating the product of step (e) with a restriction enzyme which will cleave any hybrids formed if the probe and variation(s) 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 to indicate that hybridization occurred.

Claim 12

A kit for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, which kit comprises, in packaged form, a multi container unit having: (a) one container for each oligonucleotide primer for each nucleic acid strand containing each different variation being detected, which primer(s) are substantially complementary to each strand containing each different variation, such that an extension product synthesized from one primer, when it is separated from its complement, can serve as a template for the synthesis of the extension product of the other primer so as to produce one or more amplified nucleic acid sequences if the variation(s) are present; (b) a container for an agent for polymerization; (c) a container for each of four different nucleotide triphosphates; (d) one container for each labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and (e) container(s) for reagents for detecting hybridization of the probes to the amplified sequence.

Claim 13

A kit for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids contained in a sample, which kit comprises, in packaged form, a multi container unit having: (a) one container for each oligonucleotide primer for each nucleic acid strand containing each different variation being detected, which primer(s) are substantially complementary to each strand containing each different variation, such that an extension product synthesized from one primer, when it is separated from its complement, can serve as a template for the synthesis of the extension product of the other primer so as to produce one or more amplified nucleic acid sequences if the variation(s) are present; (b) a container for an agent for polymerization; (c) a container for each of four different nucleotide triphosphates; (d) one container for each labeled sequence-specific oligonucleotide probe capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence; and (e) container(s) for reagents for detecting hybridization of the probes to the amplified sequence.

Claim 14

A kit according to claim 9, wherein said primers are oligodeoxyribonucleotides, said nucleotide triphosphates are dATP, dCTP, dGTP and TTP, the probe is labeled, and the nucleotide variation in sequence is associated with sickle cell anemia, hemoglobin C disease, the human HLA-DQa, DQβ, DRS, DPa, or DP6 region, or mitochondrial DNA.