Methods and compositions for isolating nucleic acid sequence variants
First Claim
1. A method of sorting nucleic acid molecules according to the identity of their corresponding unique nucleotide tags, said method comprising:
- (i) contacting a nucleic acid sample comprising at least two nucleic acid molecules each having a unique tag to a capture primer under hybridization conditions, wherein said capture primer is immobilized on a substrate and comprises a sequence complementary to a capture primer binding site in said at least two nucleic acid molecules, whereby said at least two nucleic acid molecules are immobilized;
(ii) contacting said immobilized at least two nucleic acid molecules to a polymerization primer under hybridization conditions, wherein said polymerization primer comprises a sequence complementary to all of or a region in the unique tag of a first of said at least two nucleic acid molecules, wherein said polymerization primer binds to the same strand of said first nucleic acid molecules as said capture primer at a location that is 5′
to said capture primer; and
(iii) isolating said first nucleic acid molecules by contacting said immobilized at least two nucleic acid molecules to a displacing nucleotide polymerase under nucleic acid polymerization conditions, wherein extension of said polymerization primer displaces said first nucleic acid molecules from said immobilized capture primer;
wherein said at least two uniquely-tagged nucleic acid molecules are sorted according to the identity of their corresponding unique nucleotide tags.
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Abstract
The invention is drawn to isolating sequence variants of a genetic locus of interest using a modified iterative primer extension method. The nucleic acids analyzed are generally single stranded and have a reference sequence which is used as a basis for performing iterative single nucleotide extension reactions from a hybridized polymerization primer. The iterative polymerization reactions are configured such that polymerization of the strand will continue if the sequence of the nucleic acid being analyzed matches the reference sequence, whereas polymerization will be terminated if the nucleic acid being analyzed does not match the reference sequence. Nucleic acid strands that have mutations can be isolated using a variety of methods and sequenced to determine the precise identity of the mutation/polymorphism. By performing the method on both strands of the nucleic acid being analyzed, virtually all possible mutations can be identified.
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Citations
20 Claims
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1. A method of sorting nucleic acid molecules according to the identity of their corresponding unique nucleotide tags, said method comprising:
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(i) contacting a nucleic acid sample comprising at least two nucleic acid molecules each having a unique tag to a capture primer under hybridization conditions, wherein said capture primer is immobilized on a substrate and comprises a sequence complementary to a capture primer binding site in said at least two nucleic acid molecules, whereby said at least two nucleic acid molecules are immobilized; (ii) contacting said immobilized at least two nucleic acid molecules to a polymerization primer under hybridization conditions, wherein said polymerization primer comprises a sequence complementary to all of or a region in the unique tag of a first of said at least two nucleic acid molecules, wherein said polymerization primer binds to the same strand of said first nucleic acid molecules as said capture primer at a location that is 5′
to said capture primer; and(iii) isolating said first nucleic acid molecules by contacting said immobilized at least two nucleic acid molecules to a displacing nucleotide polymerase under nucleic acid polymerization conditions, wherein extension of said polymerization primer displaces said first nucleic acid molecules from said immobilized capture primer; wherein said at least two uniquely-tagged nucleic acid molecules are sorted according to the identity of their corresponding unique nucleotide tags. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method of sorting nucleic acid molecules according to the presence of a unique nucleotide sequence, said method comprising:
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(i) contacting a nucleic acid sample comprising at least two nucleic acid molecules to a capture primer under hybridization conditions, wherein said capture primer is immobilized on a substrate and comprises a sequence complementary to a capture primer binding site present in said at least two nucleic acid molecules, whereby said at least two nucleic acid molecules are immobilized; (ii) contacting said immobilized at least two nucleic acid molecules to a polymerization primer under hybridization conditions, wherein said polymerization primer comprises a sequence complementary to a unique sequence present in a first of said at least two nucleic acid molecules, wherein said polymerization primer binds to the same strand of said first nucleic acid molecules as said capture primer at a location that is 5′
to said capture primer; and(iii) isolating said first nucleic acid molecules by contacting said immobilized at least two nucleic acid molecules to a displacing nucleotide polymerase under nucleic acid polymerization conditions, wherein extension of said polymerization primer displaces said first nucleic acid molecules from said immobilized capture primer; wherein said nucleic acid molecules are sorted according to the presence of said unique sequence. - View Dependent Claims (20)
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Specification