Methods of enriching for and identifying polymorphisms
First Claim
1. A process to identify in a nucleic acid sample the presence or absence of a nucleic add sequence difference with respect to a reference sequence, the process comprising:
- a) fragmenting nucleic acids in said sample;
b) linking adapter sequences to the nucleic acid fragments generated in step a;
c) binding said nucleic acid fragments to a solid support by hybridizing the adapter sequences to complementary oligonucleotides attached to said solid support to form bound nucleic acid fragments, and amplifying said bound nucleic acid fragments to produce bound and amplified fragments, whereby there is enrichment of subsets of the nucleic acid fragments; and
d) identifying the presence or absence of nucleic acid sequence differences within said bound and amplified fragments with respect to the reference sequence by hybridizing sequencing primers to the bound and amplified fragments and extending the sequencing primers.
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Abstract
The invention encompasses methods for enriching for and identifying a polymorphism within a nucleic acid sample either by separating a subset of a nucleic acid sample or by selectively replicating a subset of a nucleic acid sample such that the polymorphism is contained within a nucleic acid population with reduced complexity, and then identifying the polymorphism within the enriched nucleic acid sample. Methods also are disclosed for enriching for and identifying a polymorphism by contacting a nucleic acid sample that includes a subset of nucleic acid molecules having a sequence that binds to a sequence-specific binding activity with a molecule having a sequence-specific binding activity under conditions which permit specific binding, such that the subset of nucleic acid molecules bound to the activity is enriched for nucleic acid molecules having the sequence recognized by the sequence-specific binding activity, and detecting a polymorphism with respect to a reference sequence in the subset of nucleic acid molecules.
52 Citations
28 Claims
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1. A process to identify in a nucleic acid sample the presence or absence of a nucleic add sequence difference with respect to a reference sequence, the process comprising:
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a) fragmenting nucleic acids in said sample; b) linking adapter sequences to the nucleic acid fragments generated in step a; c) binding said nucleic acid fragments to a solid support by hybridizing the adapter sequences to complementary oligonucleotides attached to said solid support to form bound nucleic acid fragments, and amplifying said bound nucleic acid fragments to produce bound and amplified fragments, whereby there is enrichment of subsets of the nucleic acid fragments; and d) identifying the presence or absence of nucleic acid sequence differences within said bound and amplified fragments with respect to the reference sequence by hybridizing sequencing primers to the bound and amplified fragments and extending the sequencing primers. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A process to identify in a nucleic acid sample the presence or absence of a nucleic acid sequence difference with respect to a reference sequence, the process comprising:
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a) fragmenting nucleic acids in said sample; b) linking adapter sequences to the nucleic acid fragments generated in step a; c) binding said nucleic acid fragments to a solid support by hybridizing the adapter sequences to complementary oligonucleotides attached to said solid support to form bound nucleic acid fragments, and amplifying said bound nucleic acid fragments to produce bound and amplified fragments, whereby there is enrichment of subsets of the nucleic acid fragments; and d) identifying the presence or absence of nucleic acid sequence differences within said bound and amplified fragments with respect to the reference sequence by hybridizing sequencing primers to the bound and amplified fragments and ligating the sequencing primers to oligonucleotides which are also hybridized to the bound and amplified fragments. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification