Locus specific amplification using array probes

Patent metadata

Field	Value
Publication number	US-9388457-B2
Application number	US-21110008-A
Country	US
Kind code	B2
Filing date	Sep 15, 2008
Priority date	Sep 14, 2007
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

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.

First claim

What is claimed is:

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.

2. The method of claim 1 , wherein prior to step (f) amplified target nucleic acid sequences are fragmented to obtain amplified target nucleic acid fragments and the amplified target nucleic acid fragments are labeled with a detectable label to obtain labeled target nucleic acid fragments and wherein said step of analyzing comprises hybridizing the labeled target nucleic acid fragments to said array.

3. The method of claim 1 wherein said plurality of target nucleic acid sequences comprises between 1,000 and 100,000 different target nucleic acid sequences.

4. The method of claim 1 wherein said step of analyzing is to determine the genotype of polymorphisms in said target nucleic acid sequences in said nucleic acid sample.

5. The method of claim 1 wherein said step of analyzing is to determine the methylation status of one or more cytosines in said target nucleic acid sequences in said nucleic acid sample.

Assignees

• Fu Glenn K
• Shapero Michael H
• Wang Pei-Hua
• Affymetrix Inc

Inventors

• Fu Glenn K
• Shapero Michael H
• Wang Pei-Hua

Classifications

• C12Q1/6858

  Allele-specific amplification · CPC title

• C40B50/06 Primary

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title