June 1, 2006 (Vol. 26, No. 11)

Characterizing the Genome & Understanding the Hereditary Nature of Some Diseases

The genetic makeup of a human being is determined by DNA sequences that are made up of four nucleotides, adenine, cytosine, thymine, and guanine, in a multitude of combinations. Variations in these DNA sequences can alter the genetic code and result in undesirable traits or disease conditions.

Single nucleotide variations in DNA sequences, single nucleotide polymorphisms, tend to be present in or close to genetic regions that are implicated in debilitating diseases. SNPs are also stably transferred from generation to generation and thus can be used as biomarkers in population studies.

SNPs Research – Moving Ahead

The HapMap (www.hapmap.org) Project, which was established to identify genetic variations and map out the SNP pattern haplotype of the human genome, has made available a wealth of information to the SNP research community.

SNP genotyping is an exciting field. The completion of the human genome sequencing project has enabled SNP research to move forward. SNP genotyping allows for further characterization of the human genome and a better understanding of the hereditary transfer of diseases, comments Tim Harkins, Ph.D., senior product manager at Applied Biosystems(www.appliedbiosystems.com).

The study of SNPs is of great value for the medical and pharmaceutical communities as they can help predict disease association and how an individual may respond to a given drug. Human SNP genotyping has lots of implications in the context of medical significance of diseases, clinical diagnostics, improving current practice of medicine, and in drug discovery. SNP applications also extend beyond humans to agriculture, animals, plants, and fisheries for breeding and selection of desirable traits, says Zhiming Jiang, Ph.D., product manager of SNPstream at Beckman Coulter (www.beckmancoulter.com).

A good way to define SNP genotyping technologies is by their throughput. High-throughput (HT) technologies target whole genome study for discovery purposes and can handle 10,000 or more SNPs using a few hundred samples. Low-throughput (LT) technologies allow the study of 1-10 SNPs within a sample population to assess functional relevance of each particular SNP. Medium-throughput (MT) technologies fall somewhere in between.

Medium-to High-Throughput Products

Applied Biosystems offers two complete genotyping platforms. The first platform is a genotyping system using TaqMan Genotyping Assays and real-time PCR platform, including the 7900HT. This platform detects a single SNP per reaction and can be used for validation, screening, and medium SNP projects that are in the low 100s.

As part of this platform, Applied Biosystems provides an extensive collection of prevalidated assays as well as a custom assay service to support any genome, where a ready-to-use assay is generated based upon a researcher’s submission. TaqMan Genotyping Assays target 220 drug metabolism genes, and include over 2,400 validated assays.

The company recently entered into a collaboration with the Core Genotyping Facility (CGF) at SAIC-Fredrick, an NCI-funded facility. The CGF is genotyping the entire HapMap DNA panel, and Applied Biosystems is providing the subsequent haplotype analysis.

Preliminary analysis shows that using tagging SNP methods may miss important functional SNPs and rare variants. It has already been identified that there are over 40 drug metabolizing enzymatic genes that are not covered by the HapMap project. SNPBrowser version 3.5 (made available as a free software application by Applied Biosystems) uses a physical view of the genome and incorporates the HapMap project data, linkage disequilibrium maps based upon the HapMap, or Applied Biosystems’ own genotyping data, and other public information.

Researchers can use this freeware tool to design their project needs as several tagging SNP methodologies and SNP-density wizards allow researchers to identify SNPs that will meet their project needs.

Applied Biosystems’ second genotyping platform, the SNPlex Genotyping System, is a 48-plex genotyping system and is optimized for use on the 3130xl and 3730 series capillary electrophoresis instruments. SNPlex offers a complete MT-HT solution for SNP identification and characterization for genotyping applications, according to the company.

The SNPlex Genotyping System is based on oligonucleotide ligation assay/PCR technology with PCR to amplify DNA. Special dyes called Zipchute Mobility Modifiers carrying the SNP probes are used across multiple SNPlex DNA sample pools resulting in highly reproducible SNP detection. The SNPlex system can test for 12-48 SNPs simultaneously in a given sample. It can also, the company says, readily process 500-10,000 samples over a range of 100-1,000 SNPs.

While the completion of the human genome made available a large amount of information and accelerated the pace of SNP research, simultaneous improvements in SNP-genotyping technologies have also enabled rapid and easy detection of SNPs. We are seeing huge market growth in large-scale SNP genotyping studies, says Bob Pedersen, director of genomic products and product development for OpenArray products at BioTrove (www.biotrove.com).

BioTrove is targeting this exploding market with its OpenArray SNP Genotyping system based on its Through-hole technology. The OpenArray system is composed of a microscope slide-sized plate with 3,072 through-holes, equivalent to eight 384-well plates. The through-holes are further arranged in 48 sub-arrays of 64 holes.

Target SNPs of interest are preloaded into the holes for subsequent TaqMan-PCR reactions with DNA samples. Reactions are run in these holes in a 33-nL reaction volume. The hydrophobic surface chemistry coupled with the hydrophilic hole interior effectively maintains sample integrity and prevents cross-contamination among holes. Uniform liquid dispensing is enabled with the OpenArray Sample Autoloader. The OpenArray plates are thermal cycled using a standard commercial flat-block thermal cycler and read on the OpenArray NT Imager as an endpoint assay.

BioTrove provides OpenArray plates preloaded with SNP assays to researchers by reformatting TaqMan assays into the OpenArray plate. SNP genotyping that was based on traditional PCR was prohibitively expensive for large-scale genome studies. Our technology allows the traditional PCR to be used in SNP genotyping in a high-throughput flexible format and in a cost-effective manner for large-scale studies. A single researcher can generate 200,000 data points in one day using the components of the OpenArray system without use of additional robots. The range of the system is 30-3,000 SNP markers over a range of 100s to 1000s of samples, says Pedersen.

Low-Medium Throughput

Bio-Rad Laboratories (www.bio-rad.com) is targeting the group of researchers who are analyzing a particular SNP in a disease state in the low-medium throughput analysis mode.

The Bio-Rad iQ5 Real-Time PCR Detection System is based on real-time PCR technology. This system targets the lower-throughput market and can analyze up to five targets at a time. It is supported by high-quality PCR reagents, particularly the iQ Multiplex Powermix, says Hilary K. Srere, Ph.D., marketing manager, amplification technologies, gene expression division at Bio-Rad. Key SNP genotyping applications where the iQ5 is proving to be useful are gene-expression analysis, SNP detection, detection of GMOs, mutation detection, and viral-load detection.

The Bio-Plex Suspension Array system is another product that is part of a complete solution from Bio-Rad for bead-based multiplex nucleic acid analysis with extraction reagents, amplification reagents, thermocyclers, beads, the Bio-Plex instrument, and SNP Manager software.

The Bio-Plex system fulfills a need for medium-throughput SNP analysis, fitting nicely between real-time PCR (five targets) and microarrays (hundreds of targets), while still achieving a sensitivity level that allows detection of single nucleotide differences, according to Emily Dale, Ph.D., marketing manager of discovery platforms at Bio-Rad.

Bio-Plex allows analysis of up to 100 targets in one experiment with small sample requirements and offers additional flexibility as it is simple to mix and match assays as multiplex requirements change. Since up to 100 SNPs can be measured in one experiment, the reduced reagent requirements allow for a cost per SNP that is comparable to the other technologies.

A gap definitely exists in the marketplace for medium-throughput technologies that enable the validation of a small number of SNPs with large number of samples. We are targeting those researchers who have already determined their signature panel of relevant SNPs (for example 500 SNPs panel from a pool of 50,000 SNPs) for further validation and characterization in larger sample populations, says Dr. Jiang of Beckman.

Beckman serves the LT-MT market in a scalable mode with its GenomeLab CEQ and SNPstream genotyping systems respectively. The CEQ Series system enables LT study of up to 10 SNPs in a 96-well format. It is based on capillary electrophoresis technology for sequencing. The integrated software analyzes the resultant fluorescent read-outs to make the call on SNP scoring.

The GenomeLab SNPstream Genotyping System is an automated microarray-based scalable system that can process either 12 or 48 SNPs in each well of an arrayed 384-well plate. The two levels of multiplexing allow analysis of 4,600&#8211185,000 SNPs in a single plate. An automated run can handle 72 plates per run, resulting in a theoretical analysis capacity of over three million genotypes per day, according to Dr. Jiang. The SNPstream technology is based on the simple three-step primer extension chemistry that results in a fluorescent readout.

SNPstream provides a complete solution in that it has a chemistry kit to do the primer extension, fluorescent imager, image analyzer, automated plate handler, and fully integrated software that can automate the entire workflow right from run set-up to data analysis to making the genotype call, adds Dr. Jiang.

Fine Mapping and Melt Profiling

The focus of the SNP-genotyping market is currently on whole genome analysis. However, downstream there is a developing fine-mapping market that will become the next wave in SNP-genotyping research, says Harry Stylli, Ph.D., CEO of Sequenom(www.sequenom.com). We are focusing on this emerging market and are at the forefront with our MassArray system offering.

Sequenom’s MassArray system is based on mass spectrometry and determines the SNP by detecting the molecular weight of DNA fragments. Unlike other technologies, MassArray does not require any fluorescent and luminescent labels. It is based on a simple homogenous primer extension reaction in a PCR amplified target. The company owns the intellectual property rights for the use of mass spectrometry in any kind of nucleic acid analysis, according to Dr. Stylli.

All sample-preparation steps are conducted in standard microplate formats so that the end-user has maximum flexibility to configure assays for the study and also the ability to increase or reduce the number of SNPs at a later stage. Multiplexed assays are designed using dedicated assay design software. Both PCR and extension primers are ordered at the vendor of choice.

After the primer extension process the samples are spotted onto Sequenom’s SpectroChip (96-384 sample capacity) and are subject to MALDI-TOF for detection. Assays can be multiplexed with 29 data points obtained in a single reaction. Forward multiplexing capability lowers cost, increases throughput, and improves quality of data.

The hardware and software components and the test kits with all reaction ingredients makes our system offering a complete cost-effective solution to customers. We can achieve $.03 per genotype cost, which makes sample analysis affordable, says Dr. Stylli. Sequenom also offers SNP services for complete custom panel genotyping from sample preparation to final analysis.

Development of closed-tube systems, such as TaqMan, made specific SNPs analysis fast, efficient, and streamlined. While useful, the analysis of specific mutations was in itself inadequate. DNA sequencing was the logical next-step progression. Despite the drop in sequencing costs it remains expensive, and data analysis is time-consuming even with modern software tools, says Steven F. Dobrowolski, Ph.D., senior scientist at Idaho Technology (www.idahotech.com).

Idaho Technology offers two instruments based on melt-profiling technology as a fast, simple, and low-cost alternative for post-PCR SNP detection prior to the sequencing step. High-resolution melt profiling allows one to quickly triage regions where sequence variants are not present and to focus sequencing efforts only to regions containing sequence variants. Melt profiles identify regions having sequence variants and the same PCR product used for melt profiling may be recovered and subsequently used as DNA sequencing template.

This reduces turnaround time by limiting the amount of sequencing required and subsequently the amount of sequence data requiring evaluation. This is particularly important for molecular diagnostics in metabolic diseases where any delay in initiating treatment can be deleterious to the patient, explains Dr. Dobrowolski.

The company’s HR-1 Instrument is a single capillary melter with throughput of 40?? samples per hour. Its key product offering is the LightScanner system based on chemistry specifically designed for Hi-Res Melting. Hi-Res Melting is a post-PCR technique for homogenous mutation scanning and genotyping. The instrument generates high-resolution melt profiles in standard 96 or 384 microtiter plate-based formats. Intuitive software groups the results and provides automatic calling.

The LCGreen Plus Dye and the LightScanner Mastermix PCR cocktail, provided by Idaho Technology, support the reaction. The system has applications in re-sequencing and mutation discovery projects and is designed to meet the needs of high-throughput scanning projects.

Key benefits to customers using Idaho’s technology include a homogenous reaction mixture, no need for costly probes, no post-PCR processing, samples can be further evaluated by sequencing if necessary, and a highly sensitive readout is capable of detecting both heterozygous and homozygous mutations, according to Rachel Jones, product marketing manager.

Room for Improvement

While SNPs research is on the forward march, challenges still exist. Some general challenges in the SNP arena are bottlenecks in bioinformatics; assessing relevancy of information especially from data generated with whole genome SNP studies; bridging the gap between sample formats, analysis, and compatible systems; and lack of standards for comparison across different technologies, comments Dr. Jiang

Accurate genotyping results rely on the purification of high-quality contaminant-free DNA. Officials at Qiagen (www.qiagen.com) say Qiagen DNA-purification kits enable manual or automated recovery of high-quality genomic DNA from all sample types at any throughput.

If the sample quantity is limited or if many genotyping assays are to be performed, Qiagen REPLI-g kits and services, which utilize multiple displacement amplification to amplify whole genomes, are ideal, notes company spokesperson Thomas Theuringer.

Qiagen also offers a number of qualitative and quantitative PCR kits, including the HotStarTaq Plus Kit and QuantiTect Multiplex PCR Kit.

Somatic Mutations

While most SNP-analysis products focus on inherited genetic variants it has become apparent over the past two years that detecting acquired somatic mutations in tumors can also provide valuable diagnostic information. For example, a number of studies have shown that clinical response to EGFR-targeted drugs, such as Iressa or Tarceva in lung cancer, is associated with specific activating mutations within the EGFR gene.

There are significant technical challenges associated with the detection of acquired mutations in cancer due to the fact that they may be present in only a small proportion of the tumor biopsy. DxS (www.dxsgenotyping.com) says it has a number of new products to overcome this problem.

According to Stephen Little, Ph.D., CEO, the company&#8217s EGFR Mutation Test Kit combines the specificity of AstraZeneca&#8217s (www.astrazeneca.com) ARMS (allele-specific PCR) technology with the sensitivity of DxS&#8217 Scorpions signaling system to deliver a quantitative and robust validated test for EGFR mutations in tumor samples. This new approach combines a simple work flow with the ability to identify low-level genetic variation, allowing the detection of mutations not visible by conventional DNA sequencing, he explains, adding that additional products for mutations in the RAS and RAF genes will be launched later this year and that other assays are in development.

Unprecedented Demands

Many industry observers point out that system demands in the whole-genome disease association world are unprecedented. As a result, notes Illumina&#8217s (www.illumina.com) Bill Craumer, There are no simple solutions in genotyping. It&#8217s a very different environment than gene expression.

He believes that one of the most important points to understand is the degree of integration required to genotype and analyze large data setstens of thousands of samples and hundreds of thousands of markers per sample.

We believe Illumina&#8217s recent success is attributable to our ability to offer end-to-end solutions that combine: a high-density yet flexible array technology, robust and easy-to-perform assay technogies, data quality (call rates, reproducibility, Mendelian accuracy, HapMap concordance), broad genomic coverage and its close cousin, statistical/ information value per marker, says Craumer

The company has a range of SNP-genotyping offerings built around core beads-in-wells BeadArray technology (3-micron, oligo-coated beads). Some examples include the Sentrix Array Matrix and Sentrix BeadChip, the GoldenGate and Infinium assays, BeadStation and BeadLab systems, BeadStudio analysis software, and Illumina LIMS systems.

In October 2005, Illumina signed an agreement to support an NIH-funded program (PARC) with reagents and instrumentation for a study aimed at determining the impact of SNPs on individual response to statins.

Illumina can support every phase of this type of study, starting with whole-genome genotyping (Infinium assay) and subsequent phases of increasingly focused studies (Infinium and GoldenGate assays) to corral, identify, and validate causative SNPs, according to Craumer, who adds that trendwise, microsatellite-based approaches are being replaced increasingly with SNPs for linkage analysis and smaller-scale mapping studies.

Standard and Custom Panels

Last December, Affymetrix (www.affymetrix.com) launched a broad selection of standard and custom SNP panels for targeted genotyping applications. The new selection includes custom panels spanning 1,500 to 20,000 SNPs per assay, as well as seven standard SNP panels for whole-genome coding SNP analysis, disease-related studies, and common animal research.

These new SNP panels use the latest generation of highly multiplexed Molecular Inversion Probe (MIP) assay technology, giving scientists more SNP data per experiment, according to an Affymetrix spokesperson. The new GeneChip Application-Specific Fixed Assay panels include: whole-genome: 20K cSNP, 10K cSNP, 3K MALD (mapping by admixture linkage disequilibrium); disease-related candidate gene: 9K immune-inflammation panel; and animal model: 10K bovine, 5K mouse, 5K rat.

We have used an Affymetrix custom SNP panel for a study on obesity, says Michael Olivier, associate professor of physiology at the Medical College of Wisconsin. The MIP assay technology is very easy-to-use and has enabled us to complete a 1,600-sample study in less than two months with high call rates.

The new assays are the first MIP products to be released since Affymetrix acquired ParAllele BioScience last October. They are based on the same assay (formerly marketed as MegAllele) that was used successfully in the HapMap Project.

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