Locus specific amplification using array probes
First Claim
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1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, said method comprising:
- (a) fragmenting the nucleic acid sample to obtain fragments;
(b) mixing the fragments obtained in (a) with an array of probes arranged in features, wherein a feature comprises multiple copies of a target specific probe, so that fragments hybridize to complementary target specific probes to form probe;
target complexes;
(c) extending the probes in the probe;
target complexes using a polymerase, wherein the target is used as template for extension of the probe and wherein an extended probe is generated;
(d) removing the target from the extended probe by denaturation;
(e) generating a plurality of complementary copies of the extended probe, wherein said copies are not covalently attached to the surface of the solid support;
(f) allowing the copies to hybridize to the complementary target specific probes in the same feature; and
(g) analyzing the copies.
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Abstract
Methods are provided for multiplexed amplification of selected targets and analysis of the amplified targets. In preferred aspects the amplification and analysis take place on the same solid support and preferably in a localized area such as a bead or a feature of an array. In preferred aspects the analysis is a determination of sequence at one or more locations in the amplified target. The methods may be used for genotyping, sequencing and analysis of copy number.
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Citations
29 Claims
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1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, said method comprising:
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(a) fragmenting the nucleic acid sample to obtain fragments; (b) mixing the fragments obtained in (a) with an array of probes arranged in features, wherein a feature comprises multiple copies of a target specific probe, so that fragments hybridize to complementary target specific probes to form probe;
target complexes;(c) extending the probes in the probe;
target complexes using a polymerase, wherein the target is used as template for extension of the probe and wherein an extended probe is generated;(d) removing the target from the extended probe by denaturation; (e) generating a plurality of complementary copies of the extended probe, wherein said copies are not covalently attached to the surface of the solid support; (f) allowing the copies to hybridize to the complementary target specific probes in the same feature; and (g) analyzing the copies. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method for amplifying and analyzing a plurality of target sequences from a nucleic acid sample, said method comprising:
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(a) fragmenting the nucleic acid sample with a restriction enzyme to obtain fragments with known sequences at the 5′ and
3′
ends of the fragments, wherein said fragments comprise a plurality of target fragments comprising target sequences;(b) mixing the fragments obtained in (a) with a plurality of target specific splint oligonucleotides, wherein each splint oligonucleotide comprises a first target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at the 3′
end of a corresponding target fragment, and a second target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at and including the 5′
end of said corresponding target fragment, and wherein said first sequence is 5′
of said second sequence in said splint oligonucleotide, wherein target specific splint oligonucleotides further comprises a first common priming sequence at the 5′
end and a second common priming sequence at the 3′
end;(c) adding first and second primers to the mixture wherein said first primer is complementary to said first common priming sequence and said second primer is complementary to said second common priming sequence and wherein said first and second primers hybridize to said splint oligonucleotides so the first primer is adjacent to the 3′
end of the target fragment and the second primer is adjacent to the 5′
end of the target fragment;(d) ligating the first primer to the 3′
end of the target fragment and the second primer to the 5′
end of the target fragment to obtain ligated target fragments comprising a first common priming site at the 3′
end and a second common priming sites at the 5′
end;(e) after said ligating step (d) fragmenting said splint oligonucleotides; (f) amplifying the ligated target fragments from (d) to obtain amplified target fragments; and (g) analyzing the amplified target fragments by a method comprising hybridization to an array comprising a plurality of oligonucleotide probes present at known or determinable locations in the array. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification