Methods for identifying DNA copy number changes

Abstract

Methods of identifying 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.

1 . A method for estimating the copy number of a genomic region in a genomic sample comprising: 
 obtaining a sample comprising genomic DNA;    fragmenting the sample to form fragments;    reducing the complexity of the genomic DNA in the sample by amplifying a subset of the fragments;    hybridizing the amplified fragments to an array of probes wherein the array comprises a plurality of probe sets comprising at least 400,000 different oligonucleotide probes;    detecting an experimental hybridization pattern for the sample from the array;    calculating an intensity measurement for a plurality of probe sets from said experimental hybridization pattern; and    comparing the intensity measurement for a plurality of said probe sets to an intensity measurement from a reference source, to estimate the copy number of at least one genomic region in the sample relative to a reference source.    
     
     
         2 . The method of  claim 1  wherein a probe set comprises between 1 and 12 probe pairs, wherein a probe pair comprises a perfect match probe and a mismatch probe that is identical to the perfect match probe of the pair except for a mismatch position at the central position of the probe.  
     
     
         3 . The method of  claim 1  wherein the oligonucleotide probes are between 15 and 60 nucleotides.  
     
     
         4 . The method of  claim 3  wherein the oligonucleotide probes are 25 nucleotides.  
     
     
         5 . The method of  claim 1  wherein the oligonucleotide probes are 25 nucleotides and are spaced at approximately 100 base pair intervals in a predicted reduced complexity sample.  
     
     
         6 . The method of  claim 1  wherein the oligonucleotide probes of the array are spaced so that there is one probe about every 10,000 bases.  
     
     
         7 . The method of  claim 6  wherein there is one probe about every 2,500 bases.  
     
     
         8 . The method of  claim 6  wherein there is one probe about every 1,000 bases.  
     
     
         9 . The method of  claim 6  wherein there is one probe about every 100 bases.  
     
     
         10 . The method of  claim 1  wherein the at least 400,000 perfect match probes are complementary to regions of a genome that are predicted to be transcribed into mRNA.  
     
     
         11 . The method of  claim 1  wherein the step of fragmenting comprises digestion of the genomic DNA with at least one restriction enzyme  
     
     
         12 . The method of  claim 11  wherein an adaptor is ligated to the fragments and the fragments are amplified by PCR using a primer that is complementary to the adaptor.  
     
     
         13 . The method of  claim 1  wherein the reference hybridization pattern is obtained by hybridizing a matched normal control sample to the same array.  
     
     
         14 . The method of  claim 1  wherein fragmentation is by a restriction enzyme, wherein an adaptor is attached to the fragments and wherein the step of reducing the complexity is by amplification of a subset of fragments that are within a selected size range.  
     
     
         15 . The method of  claim 14  wherein the selected size range is about 400 to 2000 base pairs.  
     
     
         16 . The method of  claim 15  wherein the probes of the array are complementary to fragments that are 400 to 2000 base pairs when the sample is digested with said restriction enzyme.  
     
     
         17 . The method of  claim 16  wherein the array comprises a plurality of probe sets, wherein a probe set comprises probe pairs, wherein a probe pair is a perfect match probe that is perfectly complementary to a target and a mismatch probe that is identical to the perfect match probe except for a single base mismatch at the center of the probe.  
     
     
         18 . The method of  claim 16  wherein the probes are between 15 and 60 nucleotides.  
     
     
         19 . The method of  claim 18  wherein the probes are 25 nucleotides.  
     
     
         20 . The method of  claim 16  wherein the array comprises a plurality of genotyping probe sets, wherein a genotyping probe set comprises a plurality of perfect match probes that are complementary to a region of a target sequence that includes a polymorphic position, wherein the polymorphic position is a single nucleotide polymorphism and wherein the genotyping probe set comprises probes for each of two possible forms of a single nucleotide polymorphism.  
     
     
         21 . The method of  claim 1  wherein the perfect match probes are complementary to the genome so that there is about one probe every 10,000 bases throughout a genome.  
     
     
         22 . The method of  claim 21  wherein there about one probe every 2,500 bases.  
     
     
         23 . The method of  claim 21  wherein there is about one probe every 1,000 bases.  
     
     
         24 . The method of  claim 21  wherein there is about one probe every 100 bases.  
     
     
         25 . A method for estimating the copy number of at least one genomic region in a first biological sample, said method comprising: 
 (a) preparing a collection of nucleic acid molecules from said first biological sample;    (b) contacting said first collection of nucleic acids with a plurality of oligonucleotide probes that are complementary to the genomic region, wherein each probe is bound to a surface of a solid support at a determinable location;    (c) evaluating the binding of the collection of nucleic acid molecules to the plurality of oligonucleotide probes; and    (d) comparing the amount of binding observed to an expected amount of binding for a known copy number to estimate the copy number of the genomic region.    
     
     
         26 . The method according to  claim 25 , wherein said oligonucleotide probes comprise nucleic acids that range in size from 20 nucleotides to about 100 nucleotides in length.  
     
     
         27 . The method according to  claim 26  wherein said nucleic acids range in size from 20 nucleotides to about 40 nucleotides in length.  
     
     
         28 . The method according to  claim 26  wherein said nucleic acids range in size from 40 nucleotides to about 80 nucleotides in length.  
     
     
         29 . The method according to  claim 25  wherein said plurality of oligonucleotide probes includes sequences representative of locations distributed across at least a portion of a genome.  
     
     
         30 . The method according to  claim 29  wherein said genome is a human genome.  
     
     
         31 . The method according to  claim 29  wherein said genome is a mouse genome.  
     
     
         32 . The method according to  claim 25  wherein said locations have a uniform spacing across at least a portion of a genome.  
     
     
         33 . The method according to  claim 25  wherein said locations have a non-uniform spacing across at least a portion of a genome.  
     
     
         34 . The method according to  claim 25  wherein said plurality of oligonucleotide probes samples at least a portion of a genome at least about every 20 kilobases.  
     
     
         35 . The method according to  claim 25  wherein said plurality of oligonucleotide probes samples at least a portion of the genome at least about every 2 kilobases.  
     
     
         36 . The method according to  claim 25  wherein the nucleic acids in said collection of nucleic acids range in length from about 100 to about 10,000 nucleotides in length.  
     
     
         37 . The method according to  claim 25  wherein said collection of nucleic acids is prepared by a primer extension reaction using said biological sample as a source of genomic template DNA.  
     
     
         38 . The method according to  claim 25  wherein the expected amount of binding for a known copy number for said genomic region is obtained by contacting a second collection of nucleic acids with a plurality of oligonucleotide probes that are complementary to the genomic region, wherein the probes are the same sequence as the probes in  claim 19 , wherein the copy number of the genomic region of interest is known in the sample.  
     
     
         39 . The method according to  claim 25 , wherein the probes correspond to known coding regions.  
     
     
         40 . The method according to  claim 25 , wherein the probes correspond to polymorphic regions  
     
     
         41 . The method of  claim 25  wherein the solid support is a planar substrate.  
     
     
         42 . The method according to  claim 41  wherein the planar substrate is glass.  
     
     
         43 . The method according to  claim 41  wherein the solid support is a plurality of beads, wherein each bead carries a different oligonucleotide probe of determinable sequence.  
     
     
         44 . A method for estimating the copy number of a genomic region in a sample comprising: 
 amplifying genomic DNA from the sample;    fragmenting the amplified genomic DNA;    labeling the amplified genomic DNA;    hybridizing the fragmented, labeled amplified genomic DNA to an array of probes, wherein the array of probes comprises a plurality of probe sets wherein a probe set comprises a plurality of probes that are each complementary to different regions of a target sequence;    analyzing a resulting hybridization pattern to obtain a measurement of the signal of a probe set for a region of interest; and    comparing the signal to a control signal of known copy number to estimate the copy number of said genomic region.    
     
     
         45 . The method of  claim 44  wherein the step of amplifying reduces the complexity of the genomic DNA by preferentially amplifying a subset of the genomic DNA.  
     
     
         46 . The method of  claim 45  wherein the subset of the genomic DNA that is preferentially amplified is reproducibly amplified.  
     
     
         47 . The method of  claim 44  wherein target sequences are sequences that contain a SNP and wherein the perfect match probes of the probe set are allele specific probes that are each complementary to a region of a target sequence that contains the SNP.  
     
     
         48 . The method of  claim 44  wherein the step of amplifying is by fragmenting the sample with a restriction enzyme, attaching an adaptor, and amplifying the fragments by PCR using a single primer that is complementary to the adaptor.  
     
     
         49 . The method of  claim 44  wherein said step of amplifying is by multiple displacement amplification.  
     
     
         50 . The method of  claim 44  wherein said step of amplifying is by fragmenting, attaching common priming sequences to the fragments and amplifying the fragments.