product review
Multiple choices for SNPs Researchers face a myriad of SNP genotyping choices. Katie Cottingham
E
ven before the completion of the human genome draft sequence, analysis of single nucleotide polymorphisms (SNPs, pronounced “snips”) was heralded as the next wave for the genomics field. Knowing all the locations of single-base variations, which occur roughly once every 1000 nucleotides throughout the genome, was supposed to hold the key to curing diseases and tailoring drug therapies for individuals. Since 1999, two large consortia of private and public research entities have deposited >1.8 million SNPs into a free database. Loads of data have been generated, but where’s the payoff? “It’s not yet been proven that SNPs can be used in all the ways people forecasted that they would,” says Jim Lazar of Marligen Biosciences. Although Lazar says that research linking particular SNPs to particular medical conditions is lagging, he adds that it’s only a matter of time before reality catches up to the hype. Researchers have already found that a handful of diseases, such as sickle cell anemia, stem from a single-base change, though they caution that most disease etiologies may not be that straightforward. As Hemanth Shenoi of Promega points out, “Life is too complicated to think that everything is a single SNP.” Instead, combinations of SNPs may be important. To determine which SNPs are clinically relevant, researchers compare SNPs present in sick and healthy subjects. Sequencing is the gold standard for discovering new SNPs. However, if a researcher wants to find out which known SNPs are different between two individuals, sequencing is not typically the method of choice, according to John Butler of the National Institute of Standards and Technology. For most genotyping applications, the huge amount of data generated by sequencing is not needed to determine a one-base change. Scientists interested in SNP genotyping typically turn to methods that allow them to easily differentiate alleles, or SNP variants, without sorting through an alphabet soup of data. And there’s a dizzying number of products on the market to help scientists with the task. Table 1, which is meant to be representative rather than comprehensive, lists several SNP genotyping assays and instruments. Experts say the market is crowded, and it’s either growing or cooling off, depending on whom you ask. One company selling its technology to another is common, as is company turnover. © 2004 AMERICAN CHEMICAL SOCIETY
Genotyping technologies At first glance, each SNP genotyping method seems completely different from the next. Upon closer inspection, however, two main elements emerge. One element is that each protocol has a way of distinguishing one nucleotide from another at a particular location on a piece of DNA, either in a solution-based test or in a bead or microarray format. The second element is the detection method. SNP genotyping assays are different combinations of these elements plus a few creative tweaks, according to Pui-Yan Kwok of the University of California, San Francisco. Several SNP genotyping products differentiate alleles by hybridization. The binding of sample DNA to an oligonucleotide, or oligo, probe that is perfectly complementary to a SNP allele is detected as a match. In some assays, the subsequent dissociation or degradation of the probe is the monitored event. No enzymes are required for the oligo to bind the sample DNA, which can reduce the cost and complexity of such products. Most assays on the market, however, are based on primer extension, a process in which special enzymes and nucleotide bases are added to elongate an oligo. Primer extension assays may include a primer that is extended only if it has hybridized to the SNP region of a sample DNA. Alternatively, the oligo may be designed to hybridize to the sample DNA one base prior to the SNP. The genotype is subsequently determined by the particular base or set of bases that are extended onto the primer. M A Y 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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product review
Table 1. Selected SNP genotyping products.1 Company
Product
Product description
Affymetrix 3380 Central Expressway Santa Clara, CA 95051 www.affymetrix.com
GeneChip Human Mapping 10K Array
The 10K Array is a hybridization microarray assay that uses one universal PCR primer to genotype >10,000 SNPs/array. This reduces primer costs and makes the assay easy to use. Detection is by fluorescence.
MegaBACE SNuPe Genotyping Kit Amersham Biosciences CodeLink Human P450 SNP Bioarray 800 Centennial Ave. Piscataway, NJ 08852 www.amershambiosciences.com
SNuPe uses fluorescence to detect single-nucleotide primer extension and has a throughput of up to 13,500 SNPs/day. The CodeLink microarray assay is based on primer extension and is specifically designed for toxicogenomic screening.
Applied Biosystems 850 Lincoln Centre Dr. Foster City, CA 94404 www.appliedbiosystems.com
ABI PRISM SNaPshot Multiplex System TaqMan SNP Genotyping Assays SNPlex Genotyping System
SNaPshot system is a primer extension assay used for a few SNPs or a few samples. TaqMan products consist of a hybridization assay for interrogating a few SNPs on many samples. SNPlex system is based on OLA and can produce 400,000 genotypes in 8 h and will eventually offer >1 million genotypes/day.
Beckman Coulter, Inc. 4300 N. Harbor Blvd., Box 3100 Fullerton, CA 92834-3100 www.beckman.com
GenomeLab SNPstream Genotyping System The easily automated SNPstream system is optimized for cost-effective genotyping at any scale—from medium to very high throughput applications. Primer extension is detected by fluorescence. Less than 0.2 ng of genomic DNA is required for each genotype.
Biotage 2200 West Park Dr., Ste. 320 Westborough, MA 01581 www.biotage.com
Pyrosequencing PSQ 96 HS
Pyrosequencing is sequencing-by-synthesis. The automated system can analyze ~30,000 SNPs in 24 h using triplex reactions and is ideal for interrogating SNPs that are in close proximity to each other. One of its strengths is built-in quality control through sequence context. Almost any SNP can be analyzed.
DynaMetrix, Ltd. 48, Hyde Ave, Stotfold Herts, UK www.dynametrix-ltd.com
DASH Chemistry
DASH chemistry uses standard run conditions and can be performed in plates or on macroarrays for minimal cost. It consists of probe hybridization and a melting curve analysis. Assay design is automated via software that converts 95% of SNPs to functioning assays without optimization. Services are available.
Genteon, Inc. 8316 Miramar Mall San Diego, CA 92121 www.genteon.com
Capella 400
The Capella 400 is a CE instrument that is used to analyze unlabeled products of primer extension reactions. The instrument can test up to 3840 SNPs per individual/1-h run or 10 SNPs for 384 individuals in one run.
Illumina, Inc. 9885 Towne Centre Dr. San Diego, CA 92121 www.illumina.com
BeadStation 500GX System BeadLab Research Center Fast-Track Genotyping Services
Illumina’s assay analyzes standard products and custom projects by allelespecific extension and universal PCR, which is multiplexed at 384–1536 loci. The system uses 96- or 16-sample microarrays and features submicrometer fluorescent detection. Protocols, reagents, and software that permit confident identification of genotypes from genomic DNA are included.
OLA: oligonucleotide ligation assay 1 Some companies offer multiple instruments. Contact the vendors for their full product lines.
Methods based on other technologies are less common. Third Wave’s Invader assay uses invasive cleavage to distinguish between alleles. When two oligo probes match the sample DNA, a certain 3-D structure is formed that is recognized by a proprietary enzyme, which cleaves off a piece of one of the probes. The SNPlex system, sold by Applied Biosystems, is an oligonucleotide ligation assay in which two probes are ligated together if they are complementary to the sample sequence. Biotage’s Pyrosequencing technique is a real-time, quantitative sequencing assay. Regardless of how alleles are discriminated, the end result must be detected in some way. Almost all genotyping systems detect fluorescently labeled probes, nucleotides, or sample DNA. Genteon’s Capella CE instrument, however, has a proprietary fluorescent dye embedded in its matrix that binds to nucleic acids. Fluorescence resonance energy transfer, in which energy is transferred from one molecule to another, is used with other techniques, such as DynaMetrix’s DASH assay. Fluorescence polarization (FP) is the detection method for PerkinElmer’s 180 A
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AcycloPrime kit. In FP, large genotyping products spin at a slower rate than small products when excited by polarized light. Notable alternatives to fluorescence include chemiluminescence and MS detection. Both Biotage’s Pyrosequencing and Promega’s READIT system detect visible light signals generated from a luciferase reaction. MS is the detection method of choice for Sequenom’s MassARRAY series. Primer extension in Sequenom’s assay results in different sized products whose masses can be precisely determined by MS.
But how do you choose? Experts say that of the various permutations of allelic discrimination and detection methods, there is no one clear winner. “All commercial methods have similar accuracies,” says Kwok. Kwok and Butler agree that the selection of a SNP genotyping technology depends on the desired level of throughput, cost, availability of custom services, and flexibility. Throughput levels vary among products. Some, like the
product review
Table 1. Selected SNP genotyping products (continued).1 Company
Product
Product description
Marligen Biosciences, Inc. 2502 Urbana Pike Ijamsville, MD 21754 www.marligen.com
Signet SNP Arrays
The Signet Array kits are designed to analyze multiple SNPs on the Y chromosome and mitochondrial DNA. Marligen also develops custom SNP arrays based on allele-specific primer extension. Signet arrays are ideal for 5–500 SNPs/individual.
PerkinElmer Life & Analytical Sciences AcycloPrime-FP SNP Detection Kit 549 Albany St. Boston, MA 02188 www.perkinelmer.com
The AcycloPrime-FP primer extension assay can analyze up to 100,000 genotypes/ day and is completely scalable. Unlabeled primers are used, which drives down the cost. No transfer or washing steps are required. Detection is by fluorescence polarization.
Promega Corp. 2800 Woods Hollow Rd. Madison, WI 53711 www.promega.com
READIT SNP Genotyping System
The READIT hybridization assay is best for genotyping a few SNPs on many samples. The addition of a proprietary enzyme mastermix releases nucleotides from the ends of oligos that fully hybridize to the sample. This degradation drives a luciferase reaction that produces a light signal.
Roche Applied Science P.O. Box 50414 9115 Hague Rd. Indianapolis, IN 46250-0414 www.roche-applied-science.com
LightTyper SNP Analysis System
Roche’s LightTyper instrument monitors a solution-based melting curve analysis. Only one probe per SNP site is required for this assay. Fluorescence is used to detect the temperature at which an oligo dissociates from the sample DNA.
Sequenom 3595 John Hopkins Dr. San Diego, CA 92121 www.sequenom.com
MassARRAY System
Sequenom’s standard MassARRAY system and its benchtop MassARRAY Compact system use MALDI-TOF MS analysis. These products can also be used for other applications, such as SNP discovery, quantitative gene expression, allele frequency analysis, haplotyping, and oligonucleotide quality control.
Third Wave Technologies 502 S. Rosa Rd. Madison, WI 53719 www.twt.com
Invader Platform
The Invader assay is an invasive cleavage reaction that is detected by FRET. This assay uses standard laboratory equipment, and common reaction conditions are implemented for all DNA assays. The assay is scalable for different throughput needs.
FRET: fluorescence resonance energy transfer 1 Some companies offer multiple instruments. Contact the vendors for their full product lines.
Beckman Coulter GenomeLab SNPStream and the Third Wave Invader systems, can determine tens to hundreds of thousands of genotypes in a 24-h period. But other products are only suitable for either ultrahigh throughput (millions of genotypes per week), such as Illumina’s BeadStation, Affymetrix’s GeneChip Array, or Applied Biosystems’ SNPlex system, or low throughput (
