Methods for genotyping selected polymorphism
First Claim
1. A method for amplifying a collection of target sequences from a nucleic acid sample, the method comprising:
- fragmenting the nucleic acid sample into a plurality of fragments;
ligating one or more adaptors to the fragments to obtain adaptor-ligated fragments, wherein said adaptors comprise a first priming sequence;
contacting the adaptor-ligated fragments with probes, wherein each probe comprises a second priming sequence and a target-specific region, and the probe hybridizes to the target sequence, but not the adaptor;
extending at least some of the probes using the adaptor-ligated fragments as templates such that an extended probe comprises a sequence that is complementary to the target sequence, a sequence that is complementary to the first priming sequence and the second priming sequence; and
amplifying the extended probe with primers to said first and second priming sequences or sequences complementary to said first and second priming sequences.
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Abstract
Methods for genotyping polymorphisms using a locus specific primer that is complementary to a region near a selected polymorphism are described. Methods for synthesizing pools of locus specific primers that incorporate some degenerate positions are also disclosed. A plurality of different sequence capture probes are synthesized simultaneously using degenerate oligonucleotide synthesis. The sequence of the locus specific regions of the capture probes are related in that they have some bases that are identical in each sequence in the plurality of sequences and positions that vary from one locus specific region to another. The sequences are selected based on proximity to a polymorphism of interest and because they conform to a similar sequence pattern.
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Citations
22 Claims
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1. A method for amplifying a collection of target sequences from a nucleic acid sample, the method comprising:
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fragmenting the nucleic acid sample into a plurality of fragments; ligating one or more adaptors to the fragments to obtain adaptor-ligated fragments, wherein said adaptors comprise a first priming sequence; contacting the adaptor-ligated fragments with probes, wherein each probe comprises a second priming sequence and a target-specific region, and the probe hybridizes to the target sequence, but not the adaptor; extending at least some of the probes using the adaptor-ligated fragments as templates such that an extended probe comprises a sequence that is complementary to the target sequence, a sequence that is complementary to the first priming sequence and the second priming sequence; and amplifying the extended probe with primers to said first and second priming sequences or sequences complementary to said first and second priming sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification