Nucleic acid detection methods using universal priming
First Claim
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1. A method for determining the identification of a nucleotide at a detection position in a target sequence, comprising:
- (a) hybridizing a target sequence with a probe set to form a hybridization complex, said probe set comprising a first probe having a first portion containing an upstream universal priming site (UUP), a second portion containing a first target specific sequence and a detection position, and a second probe having a first portion containing a downsteam universal priming site (DUP) and a second portion containing a second target specific sequence, wherein said probe set further comprises at least one adapter sequence;
(b) contacting said hybridization complex with a ligase to form a ligated hybridization complex;
(c) removing non-hybridized probe sets;
(d) amplifying said ligated hybridization complex with a universal primer pair to form amplicons;
(e) immobilizing said amplicons to a solid support;
(f) contacting said immobilized amplicons with a detection label, wherein the mass of said detection label is indicative of said nucleotide at said detection position, and (g) determining a nucleotide at said detection position.
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Abstract
The present invention is directed to providing sensitive and accurate assays for genotyping with a minimum or absence of target-specific amplification.
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17 Claims
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1. A method for determining the identification of a nucleotide at a detection position in a target sequence, comprising:
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(a) hybridizing a target sequence with a probe set to form a hybridization complex, said probe set comprising a first probe having a first portion containing an upstream universal priming site (UUP), a second portion containing a first target specific sequence and a detection position, and a second probe having a first portion containing a downsteam universal priming site (DUP) and a second portion containing a second target specific sequence, wherein said probe set further comprises at least one adapter sequence;
(b) contacting said hybridization complex with a ligase to form a ligated hybridization complex;
(c) removing non-hybridized probe sets;
(d) amplifying said ligated hybridization complex with a universal primer pair to form amplicons;
(e) immobilizing said amplicons to a solid support;
(f) contacting said immobilized amplicons with a detection label, wherein the mass of said detection label is indicative of said nucleotide at said detection position, and (g) determining a nucleotide at said detection position. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification