Complexity management of genomic DNA by locus specific amplification
First Claim
1. A method of amplifying a collection of target sequences from a nucleic acid sample comprising:
- generating a collection of capture probes comprising a plurality of different species of primers wherein each species comprises a first common sequence and a 3′
variable region that is specific for a target sequence in said collection of target sequences, wherein said collection of capture probes is attached to a solid support and the 3′
end of the capture probes is available for extension;
fragmenting the nucleic acid sample;
ligating an adapter to the fragments, said adapter comprising a second common sequence;
hybridizing the adapter-ligated fragments to the collection of capture probes;
extending the capture probes; and
amplifying the extended capture probes with first and second common sequence primers.
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Accused Products
Abstract
The present invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences. In one embodiment complexity reduction can be accomplished by extension of a locus specific capture probe followed by amplification of the extended capture probe using common primers. The locus specific capture probes may be attached to a solid support. Multiple DNA sequences may be amplified simultaneously to produce a reduced complexity sample. The invention further provides for analysis of the above sample to interrogate sequences of interest such as polymorphisms. The amplified sample may be hybridized to an array, which may be specifically designed to interrogate the desired fragments for the presence or absence of a polymorphism.
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Citations
85 Claims
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1. A method of amplifying a collection of target sequences from a nucleic acid sample comprising:
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generating a collection of capture probes comprising a plurality of different species of primers wherein each species comprises a first common sequence and a 3′
variable region that is specific for a target sequence in said collection of target sequences, wherein said collection of capture probes is attached to a solid support and the 3′
end of the capture probes is available for extension;
fragmenting the nucleic acid sample;
ligating an adapter to the fragments, said adapter comprising a second common sequence;
hybridizing the adapter-ligated fragments to the collection of capture probes;
extending the capture probes; and
amplifying the extended capture probes with first and second common sequence primers. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method of amplifying a collection of target sequences from a nucleic acid sample said method comprising:
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generating a collection of capture probes comprising a plurality of different species of primers wherein each species comprises a first common sequence and a 3′
variable region that is specific for a target sequence in the collection of target sequences;
fragmenting the nucleic acid sample;
ligating an adapter to the fragments, wherein the adapter is ligated to the fragments so that the strand that is ligated to the 5′
end of the fragment strands comprises a second common sequence and the strand that is ligated to the 3′
end of the fragments lacks the complement of the second common sequence and is blocked from extension at the 3′
end;
hybridizing the adapter-ligated fragments to the collection of capture probes;
extending the capture probes; and
amplifying the extended capture probes with first and second common sequence primers. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method for genotyping one or more polymorphisms in a nucleic acid sample comprising:
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fragmenting the nucleic acid sample to generate fragments;
ligating an adaptor to the fragments said adapter comprising a first common sequence;
hybridizing a collection of capture probes to the fragments, wherein said capture probes comprise a second common sequence, a tag sequence unique for each species of capture probe, a first target specific sequence, a Type IIs restriction enzyme recognition sequence, and a second target specific sequence wherein the Type IIs restriction enzyme recognition sequence is positioned so that the enzyme will cut on the 5′
side of the polymorphic base;
extending said capture probes to generate extended capture probes;
amplifying the extended capture probes with first and second common sequence primers;
digesting the amplified product with a Type IIs restriction enzyme to generate amplified product fragments;
extending the amplified product fragments in at least one extension reaction;
hybridizing each extension reaction to an array comprising tag probes that hybridize to the tag sequences in the capture probes; and
analyzing the hybridization pattern on each of the arrays to determine at least one genotype. - View Dependent Claims (47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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76. A kit for amplifying a collection of target sequences said kit comprising:
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a collection of capture probes, wherein each species of capture probe comprises a first common sequence and a 3′
variable region that is specific for a target sequence in said collection of target sequences;
an adapter comprising a second common sequence; and
a pair of first and second common sequence primers. - View Dependent Claims (77, 78)
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79. A kit for amplifying a collection of target sequences said kit comprising:
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a collection of capture probes, wherein each species of capture probe comprises a first common sequence, a tag sequence unique for each species of capture probe, a first target specific sequence, a Type IIs restriction enzyme recognition sequence, and a second target specific sequence;
an adapter comprising a first strand comprising a second common sequence and a second strand that does not contain the complement of the second common sequence and is blocked from extension at the 3′
end; and
a pair of first and second common sequence primers. - View Dependent Claims (80, 81)
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82. A collection of capture probes attached to a solid support, wherein said solid support is selected from the group consisting of arrays, beads, microparticles, microtitre dishes and gels.
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83. A collection of capture probes, wherein each species of capture probe comprises a first common sequence, a tag sequence unique for each species of capture probe, a first target specific sequence, a Type IIs restriction enzyme recognition sequence, and a second target specific sequence, wherein the target specific sequence are specific for targets in a collection of target sequences.
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84. A method of generating a plurality of oligonucleotides comprising:
- synthesizing a plurality of oligonucleotides on a solid support and releasing the plurality of oligonucleotides from said solid support.
- View Dependent Claims (85)
Specification