Complexity management of genomic DNA by locus specific amplification
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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
111 Claims
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1-85. -85. (canceled)
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86. A method of amplifying a collection of target sequences from a nucleic acid sample, wherein each target sequence comprises a polymorphic position of interest, comprising:
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generating a collection of capture probes comprising a plurality of different species of primer sequences, wherein each species comprises a first priming sequence that is common to the capture probes in the collection and each species further comprises a 3′
target specific region that is complementary to a different target sequence in the collection of target sequences, wherein the target specific region of each capture probe hybridizes to a target sequence at a region that is between 1 and 1000 bases 3′
of a polymorphic position of interest in the target sequence, and wherein each capture probe in 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 with a restriction enzyme that cleaves the target sequences 5′
of the polymorphic position of interest;
ligating an adapter to the fragments, said adapter comprising a second priming sequence;
hybridizing the adapter-ligated fragments to the collection of capture probes, extending the capture probes through the polymorphic position in the target sequence; and
amplifying the extended capture probes with primers to said first and second priming sequences. - View Dependent Claims (87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111)
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Specification