Affymetrix, Inc.

Dendreon Corporation

Other

Affymetix, Inc.

Michael H Shapero

Keith W Jones

Glenn K Fu

Pei-Hua Wang

Stephen P A Fodor

Robert J Lipshutz

Jing Huang

Andrew Sparks

Eric Thomas Fung

Ronald J Sapolsky

Reiner Laus

Jeanette Pruzan Schmidt

Monica Chadha

Anju Shukla

Curtis L Ruegg

August Estabrook

Demao Yang

Jiang Li

Orna Mizrahi Man

Weiwei Liu

Larisa Tsavaler

Ketih W Jones

Jeremy Nathan Gollub

Dorothy Oliver

Farooq Siddiqui

Deborah Black

Lawrence Greenfield

John Efcavitch

Laurent Bellon

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.

Locus specific amplification using array probes

This disclosure provides methods and systems useful in array-based analysis of mixed nucleic acid populations, including for multiplex genotyping of a mixed nucleic acid sample and for detecting differences in copy number of a target polynucleotide and/or a target chromosome (e.g., microdeletions, duplications and aneuploidies). The disclosure also provides methods and systems useful in the diagnosis of genetic abnormalities in a mixed nucleic acid population taken non-invasively from an organism, such as a sample of blood, plasma, serum, urine stool or saliva. The disclosed methods and systems find use in multiple applications, including prenatal testing and cancer diagnostics. The disclosure is based on the hybridisation of amplified fragments from the sample, e.g. a maternal sample, which may employ molecular inversion probes MIP to an oligonucleotide array and the detection of the alleles based on different signals from the different alleles of the SNP. The disclosure also discloses how the determination of the allele ratio may be used in the determination of fetal and maternal CNVs, e.g. aneuploidies.

Array-based methods for analysing mixed samples using differently labelled allele-specific probes

This disclosure provides methods and kits useful in analysis of mixed nucleic acid populations, including for multiplex genotyping of a mixed nucleic acid sample and for detecting differences in copy number of a target polynucleotide and/or a target chromosome (e.g., microdeletions, duplications and aneuploidies). The disclosure also provides methods and systems useful in the diagnosis of genetic abnormalities in a mixed nucleic acid population taken non-invasively from an organism, such as a sample of blood, plasma, serum, urine stool or saliva. The disclosed methods and systems find use in multiple applications, including prenatal testing and cancer diagnostics. The method is based on the hybridisation of amplified fragments obtained from the sample, e.g. using molecular inversion probes (MIP) to an oligonucleotide array and the detection of the alleles based on different signals from the different alleles of the SNP.

The present invention provides for methods for discriminating between alleles at polymorphic positions in a genome. In general, the methods employ allele-specific extension of oligonucleotides that are complementary to one of the alleles at the 3' end of the oligonucleotide. The allele-specific oligonucleotides are resistant to proofreading activity from a polymerase and may be extended in an allele-specific manner by a DNA polymerase with a functional 3&#x2032; to 5&#x2032; exonuclease activity. The allele-specific oligonucleotides may be attached to a solid support such as a chip or a bead.

Methods for genotyping

Methods for genotyping polymorphisms using a locus specific primer that is complementary to a region near a selected polymorphism are described. Methods for synthesizing pools of locus specific primers that incorporate some degenerate positions are also disclosed. A plurality of different sequence capture probes are synthesized simultaneously using degenerate oligonucleotide synthesis. The sequence of the locus specific regions of the capture probes are related in that they have some bases that are identical in each sequence in the plurality of sequences and positions that vary from one locus specific region to another. The sequences are selected based on proximity to a polymorphism of interest and because they conform to a similar sequence pattern.

Methods for genotyping selected polymorphism

Methods of identifying allele-specific changes in genomic DNA copy number are disclosed. Methods for identifying homozygous deletions and genetic amplifications are disclosed. An array of probes designed to detect presence or absence of a plurality of different sequences is also disclosed. The probes are designed to hybridize to sequences that are predicted to be present in a reduced complexity sample. The methods may be used to detect copy number changes in cancerous tissue compared to normal tissue. The methods may be used to diagnose cancer and other diseases associated with chromosomal anomalies.

Methods for identifying DNA copy number changes

Methods for detecting genomic rearrangements are provided. In one embodiment, methods are provided for the use of paired end tags from restriction fragments to detect genomic rearrangements. Sequences from the ends of the fragments are brought together to form ditags and the ditags are detected. Combinations of ditags are detected by an on-chip sequencing strategy that is described herein, using inosine for de novo sequencing of short segments of DNA. In another aspect, translocations are identified by using target specific capture and analysis of the captured products on a tiling array.

Array-based translocation and rearrangement assays

The present invention provides for methods for discriminating between alleles at polymorphic positions in a genome. In general the methods employ allele specific extension of oligonucleotides that are complementary to one of the alleles at the 3' end of the oligonucleotide. The allele specific oligonucleotides are resistant to proof reading activity from a polymerase and may be extended in an allele specific manner by a DNA polymerase with a functional 3&#x2032; to 5&#x2032; exonuclease activity. The allele specific oligonucleotides may be attached to a solid support such as a chip or a bead.

Methods and kits for synthesizing a plurality of oligonucleotides are provided. Methods for providing a plurality of oligonucleotides enriched for full length oligonucleotides are provided. Truncated oligonucleotides are preferentially removed from the sample by digestion. Methods are also provided for amplification of a plurality of oligonucleotides.

Method of oligonucleotide synthesis

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Redwood City, CA, United States of America

Michael H Shapero