Reagent kit for determining specific nucleotide variations
First Claim
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1. A reagent kit for detecting the presence or absence of one or more specific nucleotides at a predetermined target position in a target nucleic-acid polymer, comprising:
- (a) a detection primer comprising a detection-primer nucleotide sequence having a primer-extension-initiation 3′
-end nucleotide which constitutes a 3′
terminal end of the detection primer, the detection-primer nucleotide sequence being complementary to a primer-hybridizing nucleotide sequence of the target nucleic-acid polymer with a nucleotide in the target nucleic-acid polymer complementary to the primer-extension-initiation 3′
-end nucleotide of the detection-primer nucleotide sequence defining a primer-end complement nucleotide, the primer-hybridizing nucleotide sequence of the target nucleic-acid polymer extending towards the 3′
end of the target polymer from the primer-end complement nucleotide, the primer-end complement nucleotide being located in the target polymer at a position 3′
-ward of the predetermined target position, the position of the primer-end complement nucleotide being subject to a constraint that no nucleotide of the same type as the one or more specific nucleotides to be detected be located in the target polymer in any position between the position of the primer-end complement nucleotide and the predetermined target position;
(b) an enzymatic polymerizing agent; and
(c) a plurality of nucleoside triphosphates including at least one deoxynucleotide and at least two different chain-terminating nucleotide analogues, at least one deoxynucleotide comprising a detectable label or an attachment moiety capable of binding a detectable label, each deoxynucleotide of said plurality of nucleoside triphosphates being complementary to a nucleotide which differs from any nucleotide to which a chain-terminating nucleotide analogue of said plurality of nucleoside triphosphates is complementary;
so that in use the detection primer can hybridize to the target nucleic-acid polymer at the primer-hybridizing nucleotide sequence and form a detection-primer extension product by an enzyme-catalyzed primer-extension reaction to permit the presence or absence of a specific nucleotide at the predetermined target position to be detected by detecting the presence or absence of a corresponding detectable label in association with the detection-primer extension product.
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Abstract
Detection of variable nucleotide(s) is based on primer extension and incorporation of detectable nucleoside triphosphates. By selecting the detection step primers from the region immediately adjacent to the variable nucleotide, this variation can be detected after incorporation of as few as one nucleoside triphosphate. Labelled nucleoside triphosphates matching the variable nucleotide are added and the incorporation of a label into the detection step primer is measured. The selection of the detection step primer is important to the method according to this invention and is dependent on the nucleotide sequence of interest. The detection step primers are preferably selected so as to span the region immediately toward the 3′ end from the variable nucleotide to be detected. The detection step primers can also be complementary to a sequence beginning several nucleotides removed from the variable nucleotide. The only limitation concerning the position of the detection step primers is that the sequence between the 3′ end of the detection step primer and the variable nucleotide to be detected must not contain a nucleotide residue of the same type as the one to be detected. The detection step primers can equally well be chosen to be complementary to either the coding or non-coding strands of the nucleotide sequence of interest.
26 Citations
24 Claims
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1. A reagent kit for detecting the presence or absence of one or more specific nucleotides at a predetermined target position in a target nucleic-acid polymer, comprising:
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(a) a detection primer comprising a detection-primer nucleotide sequence having a primer-extension-initiation 3′
-end nucleotide which constitutes a 3′
terminal end of the detection primer, the detection-primer nucleotide sequence being complementary to a primer-hybridizing nucleotide sequence of the target nucleic-acid polymer with a nucleotide in the target nucleic-acid polymer complementary to the primer-extension-initiation 3′
-end nucleotide of the detection-primer nucleotide sequence defining a primer-end complement nucleotide, the primer-hybridizing nucleotide sequence of the target nucleic-acid polymer extending towards the 3′
end of the target polymer from the primer-end complement nucleotide, the primer-end complement nucleotide being located in the target polymer at a position 3′
-ward of the predetermined target position, the position of the primer-end complement nucleotide being subject to a constraint that no nucleotide of the same type as the one or more specific nucleotides to be detected be located in the target polymer in any position between the position of the primer-end complement nucleotide and the predetermined target position;(b) an enzymatic polymerizing agent; and (c) a plurality of nucleoside triphosphates including at least one deoxynucleotide and at least two different chain-terminating nucleotide analogues, at least one deoxynucleotide comprising a detectable label or an attachment moiety capable of binding a detectable label, each deoxynucleotide of said plurality of nucleoside triphosphates being complementary to a nucleotide which differs from any nucleotide to which a chain-terminating nucleotide analogue of said plurality of nucleoside triphosphates is complementary; so that in use the detection primer can hybridize to the target nucleic-acid polymer at the primer-hybridizing nucleotide sequence and form a detection-primer extension product by an enzyme-catalyzed primer-extension reaction to permit the presence or absence of a specific nucleotide at the predetermined target position to be detected by detecting the presence or absence of a corresponding detectable label in association with the detection-primer extension product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 23)
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11. A reagent kit for detecting the presence or absence of one or more specific nucleotides at a predetermined target position in a target nucleic-acid polymer, comprising:
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(a) a detection primer comprising a detection-primer nucleotide sequence having a primer-extension-initiation 3′
-end nucleotide which constitutes a 3′
terminal end of the detection primer, the detection-primer nucleotide sequence being complementary to a primer-hybridizing nucleotide sequence of the target nucleic-acid polymer with a nucleotide in the target nucleic-acid polymer complementary to the primer-extension-initiation 3′
-end nucleotide of the detection-primer nucleotide sequence defining a primer-end complement nucleotide, the primer-hybridizing nucleotide sequence of the target nucleic-acid polymer extending towards the 3′
end of the target polymer from the primer-end complement nucleotide, the primer-end complement nucleotide being located in the target polymer at a position 3′
-ward of the predetermined target position, the position of the primer-end complement nucleotide being subject to a constraint that no nucleotide of the same type as the one or more specific nucleotides to be detected be located in the target polymer in any position between the position of the primer-end complement nucleotide and the predetermined target position;(b) an enzymatic polymerizing agent; and (c) a plurality of nucleoside triphosphates including at least one deoxynucleotide and at least one chain-terminating nucleotide analogue, at least one chain-terminating nucleotide analogue comprising a detectable label or an attachment moiety capable of binding a detectable label, each deoxynucleotide of said plurality of nucleoside triphosphates being complementary to a nucleotide which differs from any nucleotide to which a chain-terminating nucleotide analogue of said plurality of nucleoside triphosphates is complementary; so that in use the detection primer can hybridize to the target nucleic-acid polymer at the primer-hybridizing nucleotide sequence and form a detection-primer extension product by an enzyme-catalyzed primer-extension reaction to permit the presence or absence of a specific nucleotide at the predetermined target position to be detected by detecting the presence or absence of a corresponding detectable label in association with the detection-primer extension product. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24)
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Specification
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Current AssigneeOrchid Cellmark Incorporated (Laboratory Corporation Of America Holdings)
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Original AssigneeOrchid Cellmark Incorporated (Laboratory Corporation Of America Holdings)
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InventorsSöderlund, Hans E., Syvanen, Anne-Christine
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Primary Examiner(s)Myers, Carla J.
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Application NumberUS08/465,322Publication NumberTime in Patent Office4,173 DaysField of Search435/5, 435/6, 435/91.2, 435/91.5, 435/91.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 935/8, 935/16, 935/78US Class Current435/6.11CPC Class CodesC12Q 1/6834 Enzymatic or biochemical co...C12Q 1/6869 Methods for sequencingC12Q 1/6886 for cancer immunoassay for ...C12Q 2535/101 Sanger sequencing method, i...C12Q 2535/125 Allele specific primer exte...C12Q 2563/101 radioactivity, e.g. radioac...
