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.