Locus specific amplification using array probes
First Claim
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1. A method for amplifying a plurality of target nucleic acid sequences from an un-amplified nucleic acid sample and analyzing the amplified target sequences, said method comprising:
- (a) fragmenting the un-amplified nucleic acid sample to obtain un-amplified target nucleic acid fragments;
(b) mixing the un-amplified target nucleic acid 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 un-amplified target nucleic acid 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 un-amplified target nucleic acid fragment is used as template for extension of the probe and wherein an extended probe is generated;
(d) removing the un-amplified target nucleic acid fragment 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, wherein the plurality of target nucleic acid sequences comprises between 100 and 100,000 different target nucleic acid sequences.
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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
5 Claims
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1. A method for amplifying a plurality of target nucleic acid sequences from an un-amplified nucleic acid sample and analyzing the amplified target sequences, said method comprising:
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(a) fragmenting the un-amplified nucleic acid sample to obtain un-amplified target nucleic acid fragments; (b) mixing the un-amplified target nucleic acid 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 un-amplified target nucleic acid 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 un-amplified target nucleic acid fragment is used as template for extension of the probe and wherein an extended probe is generated;(d) removing the un-amplified target nucleic acid fragment 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, wherein the plurality of target nucleic acid sequences comprises between 100 and 100,000 different target nucleic acid sequences. - View Dependent Claims (2, 3, 4, 5)
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Specification