Locus specific amplification using array probes

• US 9,388,457 B2
• Filed: 09/15/2008
• Issued: 07/12/2016
• Est. Priority Date: 09/14/2007
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fu, Glenn K., Shapero, Michael H., Wang, Pei-Hua
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US12/211,100
• Publication Number:
  US 20090117573A1
• Time in Patent Office: 2,857 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

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.

Claims

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.