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Genotyping Single Nucleotide Polymorphisms

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Genotyping Single Nucleotide Polymorphisms REVIEWS ACCESSING GENETIC VARIATION: GENOTYPING SINGLE NUCLEOTIDE POLYMORPHISMS Ann-Christine Syvänen Understanding the relationship between genetic variation and biological function on a genomic scale is expected to provide fundamental new insights into the biology, evolution and pathophysiology of humans and other species. The hope that single nucleotide polymorphisms (SNPs) will allow genes that underlie complex disease to be identified, together with progress in identifying large sets of SNPs, are the driving forces behind intense efforts to establish the technology for large-scale analysis of SNPs. New genotyping methods that are high throughput, accurate and cheap are urgently needed for gaining full access to the abundant genetic variation of organisms. LINKAGE DISEQUILIBRIUM Comparison of genomic DNA sequences in different These SNPs are useful as markers in population 6,7 MAPPING individuals reveals some positions at which two, or in genetics and evolutionary studies . Analysing single nucleotide some cases more than two, bases can occur. These sin- The reason for the current enormous interest in polymorphism alleles in gle nucleotide polymorphisms (SNPs) are highly SNPs is the hope that they could be used as markers to population-based studies to identify loci that are associated abundant, and are estimated to occur at 1 out of every identify genes that predispose individuals to common, 1,2 with a particular disease or 1,000 bases in the human genome . Depending on multifactorial disorders by using LINKAGE DISEQUILIBRIUM phenotype. where a SNP occurs, it might have different conse- (LD) MAPPING8,9. The rationale would be to genotype a quences at the phenotypic level. SNPs in the coding collection of SNPs that occur at regular intervals and regions of genes that alter the function or structure of cover the whole genome to detect genomic regions in the encoded proteins are a necessary and sufficient which the frequencies of the SNP alleles differ between cause of most of the known recessively or dominantly patients and controls. It is assumed that the SNP alleles inherited monogenic disorders. These SNPs are rou- are inherited together with the disease-predisposing tinely analysed for diagnostic purposes. Another alleles through the generations because they are physi- important group of SNPs are those that alter the pri- cally close to each other. The disease-predisposing mary structure of a protein involved in drug metabo- genes could then be localized and isolated, and pro- lism. These SNPs are targets for pharmacogenetic teins encoded by them would be valuable targets for analyses3. Missense SNPs in the coding regions of developing new therapeutic drugs. As a result of the genes, such as the two SNPs in the apolipoprotein E efforts of the SNP Consortium — a collaboration of gene4 and the factor V Leiden mutation5, can also con- 14 major pharmaceutical companies and the Department of tribute to common disease. This type of SNP can be Wellcome Trust, as well as members of the Human Medical Sciences — analysed to assess the risk of an individual for a par- Genome Project1 — there are almost 2 million SNPs in Molecular Medicine, ticular disease. In addition, it is likely that SNPs in the public databases, and perhaps twice that number of Uppsala University, regulatory regions of genes might influence the risk of SNPs in commercial databases, such as that of Celera University Hospital, 2 75185 Uppsala, Sweden. common disease. However, most SNPs are located in Genomics . The SNPs are now being verified experi- e-mail: Ann-Christine. non-coding regions of the genome, and have no direct mentally, and their distributions between and within [email protected] known impact on the phenotype of an individual. populations are being assessed10,11. 930 | DECEMBER 2001 | VOLUME 2 www.nature.com/reviews/genetics © 2001 Macmillan Magazines Ltd REVIEWS Table 1 | Recent or large studies involving SNP genotyping Purpose of study No. of No. of No. of Method SNPs samples genotypes SNPs in the ACE gene131 13 1,300 17,000 Restriction-site analysis NOD2 gene in Crohn disease132 13 1,300 17,000 Invader115 and TaqMan50 assays Mapping complex disease traits 109 299 33,000 Allele-specific PCR60 in mice34 Sequence diversity of 20 2,200 44,000 Oligonucleotide ligation assay95 the APOE gene133 APOE haplotypes in 60 1,000 60,000 TaqMan assay50 Alzheimer disease32 Ancestral alleles of 214 412 99,000 GeneChip39 human SNPs7 Carrier frequencies of 31 4,400 140,000 Primer extension on microarrays82 disease mutations134 Haplotype tagging in 122 1,500 180,000 Invader PCR assay115 diabetes candidate genes135 Linkage disequilibrium 312 681 210,000 Multiplex primer extension69,71,137 in the 5q31 region136 ACE, angiotensin I-converting enzyme (peptidyl-dipeptidase A)1; APOE, apolipoprotein E; SNP, single nucleotide polymorphism. But how many SNPs are needed? Although recent these routine applications is now available20, although studies indicate that LD is structured into discrete these fields would obviously benefit from lower genotyp- blocks in the human genome12,13, the range and distrib- ing costs. TABLE 1 gives some examples of recent or large ution of LD in different populations is largely unknown. studies in which SNP genotyping had a fundamental role. Furthermore, the effect of genotype on disease pheno- The scale of these projects is modest, and it is certain that type varies between disorders and populations owing to larger studies that have not been published have been genetic and environmental heterogeneity14. For these conducted, or are in progress, in the commercial sector. reasons, it is difficult to estimate the number of SNPs or Key targets to improve SNP genotyping technology the number of samples that would be required for a suc- are cost, simplicity of assay design, throughput and cessful genome-wide LD study. The estimates that have accuracy. One approach for increasing throughput is to been made vary widely, from 1 million15 or 0.5 million16 devise automated assay formats of the generally used to as little as 30,000 SNPs17. If 0.5 million SNPs were to reaction principles for genotyping individual SNPs, and be analysed in, for example, 1,000 individuals, and if the to use a ‘brute force’ strategy by multiplying these auto- project was to be carried out in a year, ~1.5 million SNP mated platforms. This strategy is analogous to the genotypes per day would be produced. At genotyping implementation of Sanger’s dideoxy method for DNA costs as low as US 10 cents per SNP,this example project sequencing21 by the Human Genome Project2,22.A sec- would cost US $50 million. The throughput required ond approach to increase throughput is to multiplex the for this project, which comprises only a modest number biochemical genotyping reactions, instead of the plat- of samples, is about 100-fold greater than the capacity of forms. Microarray technology is a typical example of SNP-genotyping technology available now. Both the this approach. The most challenging approach is to costs and the throughput would prohibit executing the develop completely new molecular strategies for SNP project in even the largest genotyping centres. For typ- genotyping. In this article, I review the current state of ing 30,000 SNPs in 1,000 individuals in a year, about a the art of SNP-genotyping technology, with an empha- tenfold increase in the capabilities of current technology sis on how amenable the different methods are to multi- would be enough. plexing to increase throughput and bring down the A more feasible alternative to random whole-genome costs of the assays. Several promising new principles and SNP mapping is to use SNP markers in or close to candi- assay formats also discussed. date genes, or in candidate genomic regions. For example, the genotyping of 20 SNPs in 50 candidate genes (1,000 Principles of SNP-genotyping methods SNPs) or of 1 SNP per 10 kb in a 10-million-bp candidate The ability of hybridization with allele-specific genomic region in 1,000 samples represents a reasonable oligonucleotides (ASO) to detect a single base mis- goal of 1 million genotypes. It would be possible to carry match was first shown in 1979 (REF. 23), and then used out a project of this size within a few months in an inter- to detect the sickle-cell mutation in the β-globin gene mediate-sized genotyping centre equipped with modern by Southern blot hybridization to human genomic SNP-genotyping technology. For diagnosis and carrier DNA in 1983 (REF. 24). Identification of a single base screening of monogenic disorders and for routine phar- change in the 6 × 109 bp of the diploid human genome macogenetic applications, tens of SNPs are typically is, however, a demanding task. Not until the PCR tech- analysed in several thousand samples3,18,19. SNP-genotyp- nique was invented25,26, did it become possible to ing technology with acceptable throughput and cost for design useful assays for genotyping SNPs in complex NATURE REVIEWS | GENETICS VOLUME 2 | DECEMBER 2001 | 931 © 2001 Macmillan Magazines Ltd REVIEWS REVERSE DOT BLOT genomes. BOX 1 summarizes six central biochemical PCR technique was developed further by several A genotyping method based on reaction principles that underlie SNP-genotyping groups to allow allele-specific amplification and geno- hybridization between allele- methods, and FIG. 1 illustrates how some of the current typing of SNPs31. The use of ASOs as hybridization specific oligonucleotide probes SNP-genotyping platforms have been devised by com- probes or as PCR primers is the basis for the SNP- that have been immobilized on a membrane, and amplified bining a reaction principle with an assay format and a genotyping assays that are referred to as ‘homogeneous’, DNA fragments in solution. detection strategy. because they contain no separation steps and are moni- Some of the early SNP-genotyping assays used for tored in real time during PCR.
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