Length determination of nucleic acid repeat sequences by discontinuous primer extension
First Claim
1. A method for determining the number of repeat units in a repeat region of a target nucleic acid comprising the steps of:
- (A) contacting a plurality of different-sequence primers with a polynucleotide sample under conditions effective for said primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein either (1) each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment, or (2) one or more polynucleotides in the sample are tagged polynucleotides that contain a tag segment having a nucleotide sequence that uniquely identifies the attached polynucleotide;
(B) performing a first primer extension reaction on said hybrid(s) using a first primer extension reagent;
(C) separating the target-primer hybrid(s) and unreacted first primer extension reagent;
(D) performing a second primer extension reaction on said hybrid(s) using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a photodestructible label attached thereto;
(E) separating the target-primer hybrid(s) from unreacted second primer extension reagent;
(F) measuring a signal produced by the label;
(G) treating the label with light to destroy a signal-producing property of the label so as to render the label undetectable; and
(H) repeating a cycle of steps (A) through (G) until the signal detected in the target-primer hybrid(s) is substantially less than a signal detected in a previous cycle; and
(I) prior to step (F), at least an aliquot of either (1) said different-sequence primers or (2) said tagged sample polynucleotides are contacted with an addressable array of immobilized, different tag complements, and each different tag complement contains a sequence that is complementary to one of said tag segments, under conditions effective to hybridize the tag segments to corresponding tag complements on the support.
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Abstract
Disclosed is a method for determining the number of repeat units in a repeat region of a target nucleic acid. In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle.
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Citations
27 Claims
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1. A method for determining the number of repeat units in a repeat region of a target nucleic acid comprising the steps of:
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(A) contacting a plurality of different-sequence primers with a polynucleotide sample under conditions effective for said primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein either (1) each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment, or (2) one or more polynucleotides in the sample are tagged polynucleotides that contain a tag segment having a nucleotide sequence that uniquely identifies the attached polynucleotide; (B) performing a first primer extension reaction on said hybrid(s) using a first primer extension reagent; (C) separating the target-primer hybrid(s) and unreacted first primer extension reagent; (D) performing a second primer extension reaction on said hybrid(s) using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a photodestructible label attached thereto; (E) separating the target-primer hybrid(s) from unreacted second primer extension reagent; (F) measuring a signal produced by the label; (G) treating the label with light to destroy a signal-producing property of the label so as to render the label undetectable; and (H) repeating a cycle of steps (A) through (G) until the signal detected in the target-primer hybrid(s) is substantially less than a signal detected in a previous cycle; and (I) prior to step (F), at least an aliquot of either (1) said different-sequence primers or (2) said tagged sample polynucleotides are contacted with an addressable array of immobilized, different tag complements, and each different tag complement contains a sequence that is complementary to one of said tag segments, under conditions effective to hybridize the tag segments to corresponding tag complements on the support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for determining the number of repeat units in a repeat region of a target nucleic acid comprising the steps of:
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(A) contacting a plurality of different-sequence primers with a polynucleotide sample under conditions effective for said primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein either (1) each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment, or (2) one or more polynucleotides in the sample are tagged polynucleotides that contain a tag segment having a nucleotide sequence that uniquely identifies the attached polynucleotide, (B) performing a first primer extension reaction on said hybrid(s) using a first primer-extension reagent, wherein the first primer extension reagent includes an unlabeled extendible nucleotide; (C) separating the target-primer hybrid(s) from unreacted first primer extension reagent; (D) performing a second primer extension reaction on said hybrid(s) using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto, (E) separating the target-primer hybrid(s) from unreacted second primer extension reagent and unreacted primer termination reagent; (F) measuring a signal produced by the label; (G) repeating a cycle of steps (A) through (F) until a signal is detected indicating incorporation of the nucleotide terminator; and (H) prior to step (F), at least an aliquot of either (1) said different-sequence primers or (2) said tagged sample polynucleotides are contacted with an addressable array of immobilized, different tag complements, and each different tag complement contains a sequence that is complementary to one of said tag segments, under conditions effective to hybridize the tag segments to corresponding tag complements on the support. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification