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Methods and Compositions for Whole Genome Amplification and Genotyping

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Methods and Compositions for Whole Genome Amplification and Genotyping (19) & (11) EP 2 264 188 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 22.12.2010 Bulletin 2010/51 C12Q 1/68 (2006.01) (21) Application number: 10175210.3 (22) Date of filing: 17.06.2004 (84) Designated Contracting States: • Steemers, Frank AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Encinitas, CA 92024 (US) HU IE IT LI LU MC NL PL PT RO SE SI SK TR (74) Representative: Murphy, Colm Damien et al (30) Priority: 20.06.2003 US 600634 Ipulse 08.10.2003 US 681800 26 Mallinson Road London (62) Document number(s) of the earlier application(s) in SW11 1BP (GB) accordance with Art. 76 EPC: 04776759.5 / 1 636 337 Remarks: This application was filed on 03-09-2010 as a (71) Applicant: Illumina, Inc. divisional application to the application mentioned San Diego, California 92121-1975 (US) under INID code 62. (72) Inventors: • Gunderson, Kevin Encinitas, CA 92024 (US) (54) Methods and compositions for whole genome amplification and genotyping (57) There is disclosed a method for indicating the presence of single nucleotide polymorphisms (SNPs) of interest in typable loci of a genome, comprising the steps of: (a) representationally amplifying a native genome to ob- tain a representative population of genome fragments comprising said typable loci; (b) contacting said fragments with an array of different immobilized nucleic acid probes under conditions where- in probe-fragment hybrids are formed, wherein said fragments have a concentration of at least 1 Pg/ul of DNA and wherein said immobilized nucleic acid probes comprise a region that is complementary to typable loci of a ge- nome; (c) contacting said probe-fragment hybrids with a modi- fication enzyme, whereby if the typable locus of a fragment contains a SNP of interest, a detection moiety is attached to the hybridized probe; and (d) detecting the probe, thereby indicating the presence of said single nucleotide polymorphisms of interest at typable loci of said genome. EP 2 264 188 A1 Printed by Jouve, 75001 PARIS (FR) 1 EP 2 264 188 A1 2 Description sociation studies. A major limitation to whole genome association studies is the lack of a technology to perform FIELD OF THE INVENTION highly-multiplexed SNP genotyping. The generation of the complete haplotype map of the human genome [0001] The present invention relates generally to ge- 5 across major ethnic groups will provide the SNP content netic analysis and more specifically to amplification of for whole genome association studies (estimated at whole genomes and genotyping based on pluralities of about 200,000-300,000 SNPs). However, currently avail- genetic markers spanning genomes. able genotyping methods are cumbersome and ineffi- cient for scoring the large numbers of SNPs needed to BACKGROUND OF THE INVENTION 10 generate a haplotype map. [0006] Thus there is a need in the art for methods of [0002] Most of any one person’s DNA, some 99.9 per- simultaneously interrogating large numbers of gene loci cent, is exactly the same as any other person’s DNA. on a whole genome scale. Such benefits will affect the The roughly 0.1% difference in the genome sequence genomic discovery process and the genetic analysis of accounts for a wide variety of the differences among peo- 15 diseases, as well as the genetic analysis of individuals. ple, such as eye color and blood group. Genetic variation This invention satisfies this need and provides other ad- also plays a role in whether a person is at risk for getting vantages as well. This invention describes and demon- particular diseases or whether a person is likely to have strates a method to perform large scale multiplexing re- a favorable or adverse response to a particular drug. Sin- actions enabling a new era in genomics. gle gene differences in individuals have been associated 20 with elevated risk for acquiring a variety of diseases, such SUMMARY OF THE INVENTION as cystic fibrosis and sickle cell disease. More complex interrelationships among multiple genes and the environ- [0007] In one aspect, the present invention features a ment are responsible for many traits like risk for some method of detecting one or several typable loci contained common diseases, such as diabetes, cancer, stroke,25 within a given genome, where the method includes the Alzheimer’s disease, Parkinson’s disease, depression, steps of providing an amplified representative population alcoholism, heart disease, arthritis and asthma. of genome fragments having such typable loci, contact- [0003] Genetic-based diagnostic tests areavailable for ing the genome fragments with a plurality of nucleic acid several highly penetrant diseases caused by single probes having sequences corresponding to the typable genes, such as cystic fibrosis. Such tests can be per- 30 loci under conditions wherein probe- fragment hybrids are formed by probing for particular mutations or polymor- formed; and detecting typable loci of the probe- fragment phisms in the respective genes. Accordingly, risk for con- hybrids. In particular embodiments these nucleic acid tracting a particular disease can be determined well be- probes are at most 125 nucleotides in length. However, fore symptoms appear and, if desired, preventative probes having any of a variety of lengths or sequences measures can be taken. However, it is believed that the 35 can be used as set forth in more detail below. majority of diseases, including many common diseases [0008] In another aspect, the present invention fea- such as diabetes, heart disease, cancers, and psychiatric tures a method of detecting typable loci of a genome disorders, are affected by multiple genes as well as en- including the steps of providing an amplified represent- vironmental conditions. Thus, diagnosis of such diseases ative population of genome fragments that has such typa- based on genetics is considerably more complex as the 40 ble loci, contacting the genome fragments with a plurality number of genes to be interrogated increases. of nucleic acid probes having sequences corresponding [0004] Recently, through a variety of genotyping ef- to the typable loci under conditions wherein probe-frag- forts, a large number of polymorphic DNA markers have ment hybrids are formed; and directly detecting typable been identified, many of which are believed to be asso- loci of the probe-fragment hybrids. ciated with the probability of developing particular traits 45 [0009] In a further aspect, the present invention fea- such as risk of acquiring known diseases. Exemplary pol- tures a method of detecting typable loci of a genome ymorphic DNA markers that are available include single including the steps of providing an amplified represent- nucleotide polymorphisms (SNPs) which occur at an av- ative population of genome fragments having the typable erage frequency of more than 1 per kilobase in human loci; contacting the genome fragments with a plurality of genomic DNA. Many of these SNPs are likely to be ther- 50 immobilized nucleic acid probes having sequences cor- apeutically relevant genetic variants and/or involved in responding to the typable loci under conditions wherein genetic predisposition to disease. However, current immobilized probe-fragment hybrids are formed; modi- methods for genome- wide interrogation of SNPs and oth- fying the immobilized probe-fragment hybrids; and de- er markers are inefficient, thereby rendering the identifi- tecting a probe or fragment that has been modified, there- cation of useful diagnostic marker sets impractical. 55 by detecting the typable loci of the genome. [0005] The ability to simultaneously genotype large [0010] The invention also provides a method, including numbers of SNP markers across a DNA sample is be- the steps of (a) providing a plurality of genome fragments, coming increasingly important for genetic linkage and as- wherein the plurality of genome fragments has at least 2 3 EP 2 264 188 A1 4 100 ug of DNA having a complexity of at least 1 Giga- [0016] Further provided is a methodincluding the steps bases; (b) contacting the plurality of genome fragments of (a) contacting a plurality of genome fragments with a with a plurality of different immobilized nucleic acid plurality of different immobilized nucleic acid probes un- probes, wherein at least 500 of the different nucleic acid der conditions wherein immobilized probe-fragment hy- probes hybridize with genome fragments to form probe- 5 brids are formed; (b) modifying the immobilized probes fragment hybrids; and (c) detecting typable loci of the while hybridized to the genome fragments, thereby form- probe-fragment hybrids. ing modified immobilized probes; (c) removing said ge- [0011] A method of the invention can also include the nome fragments from said probe-fragment hybrids; and steps of (a) providing a plurality of genome fragments, (d) detecting the modified immobilized probes after re- wherein the plurality of genome fragments has a concen- 10 moving the genome fragments, thereby detecting typable tration of at least 1 ug/ul of DNA having a complexity of loci of the genome fragments. at least 1 Gigabases; (b) contacting the plurality of ge- [0017] The invention also provides a method including nome fragments with a plurality of different immobilized the steps of (a) representationally amplifying a native ge- nucleic acid probes, wherein at least 500 of the different nome, wherein an amplified representative population of nucleic acid probes hybridize with genome fragments to 15 genome fragments having the typable loci is produced form probe-fragment hybrids; and (c) detecting typable under isothermal conditions; (b) contacting the genome loci of the probe-fragment hybrids. fragments with a plurality of nucleic acid probes having [0012] In an additional aspect, the present invention sequences corresponding to the typable loci under con- features a method of amplifying genomic DNA, including ditions wherein probe-fragment hybrids are formed; and the steps of providing isolated double stranded genomic 20 (c) detecting typable loci of the probe-fragment hybrids.
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