# US 2010/0167295 A1 · Methods for haplotyping genomic dna > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0167295 A1 - Document ID: US20100167295A1 - Status: Published application - Kind: A1 - Application number: 12/569,044 - Filed: 2009-09-29 - Published: 2010-07-01 - Earliest claimed: 2003-05-12 - Assignees: Fred Hutchinson Cancer Research Center; Institute for Systems Biology - Inventors: Effie W. Petersdorf; Zhen Guo; Leroy Hood - Web page: https://patentgrep.com/patent/US20100167295A1 ## CPC - C12Q 1/6813: Hybridisation assays - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention provides a novel method for specifically isolating and separating large segments of genomic DNA that can subsequently be used to determine a genomic haplotype. The invention relies on using a solid phase having a flat surface arrayed with oligonucleotides designed to specifically hybridize to each particular haplotype of an individual sample, e.g., oligonucleotides designed to specifically hybridize with each of the two HLA-B haplotypes, HLA-A, HLA-C, HLA-DR, HLA-DQ, and the like. The genomic DNA is contacted and hybridized to the arrayed oligonucleotides to form a genomic DNA/oligonucleotide complex. The excess genomic DNA is washed away and the haplotype separated genomic DNA is denatured from the oligonucleotide probe and collected. The method of the present invention allows for the separation of genomic DNA fragments of between approximately 2 to about 4 megabases (Mb). Separation of the haplotypes of large genomic DNA fragments allows for linkage analysis of other HLA alleles and polymorphisms, microsatellite, SNPs, and the like across a large span of the HLA region, including HLA-A, -B, -C, and HLA-DRB1 regions. This linkage analysis is particularly useful when HLA typing for an individual with limited family HLA typing available. ## Claims ### Claim 1 A method for separating fragments of genomic DNA by haplotype, comprising: (a) providing a solid surface comprising covalently bound oligonucleotide probes having a nucleotide sequence complementary to each haplotype of an allele; (b) contacting the oligonucleotide probe with a sample of genomic DNA under conditions conducive to specific hybridization of the genomic DNA to the oligonucleotide probes to form a genomic DNA/oligonucleotide complex; (c) removing excess genomic DNA; (d) denaturing the genomic DNA/oligonucleotide complex; and (e) separately collecting the genomic DNA corresponding to each haplotype; thereby separating the fragments of genomic DNA by haplotype. ### Claim 2 (canceled) ### Claim 3 (depends on 1) The method according to claim 1, wherein the oligonucleotide probes are about 20 to about 40 bases in length. ### Claim 4 (depends on 1) The method according to claim 1, wherein the oligonucleotide probes are spaced to allow independent manipulation of the genomic DNA hybridized to each probe. ### Claim 5 (depends on 1) The method of claim 1, wherein the hybridization is carried out at room temperature overnight. ### Claim 6 A method for determining cis linkage of alleles to an HLA locus haplotype, comprising: (a) providing a solid surface comprising a covalently bound plurality of oligonucleotide probes having a nucleotide sequence complementary to said HLA locus haplotype; (b) contacting the oligonucleotide probes with a sample of genomic DNA under conditions conducive to specific hybridization of the genomic DNA to the oligonucleotide probes to form a genomic DNA/oligonucleotide complex; (c) removing excess genomic DNA; (d) denaturing the genomic DNA/oligonucleotide complex; (e) separating the genomic DNA by haplotype; (f) amplifying selectively a region of interest in the haplotype separated genomic DNA using asymmetric PCR and labeled primers to form a labeled, single stranded DNA sample; (g) contacting the labeled, single stranded DNA sample with a microarray comprising a plurality of oligonucleotide probes under conditions conducive to hybridization; and (i) detecting a hybridization pattern for the DNA sample and assigning an allele type with cis linkage to the HLA locus haplotype of the genomic DNA sample. ### Claim 7 (depends on 6) The method of claim 6, wherein the plurality of oligonucleotide probes is selected to be specific for the haplotypes of the HLA-B locus. ### Claim 8 (canceled) ### Claim 9 (depends on 1) The method of claim 1, wherein the hybridization is carried out at a temperature of less than 30 degrees Celsius for 3 hours or more. ### Claim 10 (depends on 1) The method according to claim 1, wherein the solid surface comprises glass. ### Claim 11 (depends on 1) The method according to claim 1, wherein the solid surface is flat. ### Claim 12 (depends on 1) The method according to claim 1, wherein the solid surface is a bead. ### Claim 13 (depends on 1) The method according to claim 1, wherein the solid surface has been derivatized with a vapor phase deposition of an aminoalkyltrialkoxysilane. ### Claim 14 (depends on 13) The method according to claim 13, wherein the aminoalkyltrialkoxysilane is 3-aminopropyltrimethoxylsilane. ### Claim 15 (depends on 1) The method according to claim 1, wherein said covalently bound oligonucleotide probes further comprise a linking group, and wherein said linking group is sufficient to provide optimum binding of said fragments of genomic DNA to said oligonucleotide probes. ### Claim 16 (depends on 1) The method according to claim 1, wherein said removing step is carried out by washing with buffer. ### Claim 17 (depends on 15) The method according to claim 15, wherein said linking group is a poly dT spacer.