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OMICS Sample Prep Continued from page 29 At the Knowledge Foundation conference he will describe work undertaken in collabora- tion with Hoffman LaRoche (www.roche. com) using these springboard-like nanome- chanical sensors to specifically detect small RNA from serum using cantilever array sen- sors within 10–15 minutes. "You have a sample from cells, you lyse the cells, then you sediment the debris and inject the supernatant." Because miRNA in blood may be present at concentrations of up to 200 million per milliliter and quite stable (as opposed to its longer RNA cousins), it should in principle be quite easy to measure, "This is not some- thing where you're going for single molecule detection," he notes. The sensors are coated with matching se- quences which detect bound complementary miRNA in a couple of ways, with no labeling or modification of the target necessary, either by bending of bar, or by changing the bar's oscillation. The technology was initially de- rived from scanning probe microscopy, and delivers sensitivity at the Angstrom level that can be read out using laser optics. Any kind of small mechanical sensor will react to its environment and so it's manda- tory to include reference sensors "which are able to deconvolute any kind of background nonspecific binding from the real signal we are looking at," explains Dr. Hegner. "We As part of Eureka Genomics' Mass Genotyping by Sequencing Technology methodology, DNA is heated to melt it apart and break it into smaller pieces, which makes the hybridization of the next set of probes onto it much easier. always have a minimum of two sensors. It's a differential readout." The team is also collaborating with the California Institute of Technology to devel- op a version using integrated nanoelectron- ics in the springboard itself "where we don't need an optical readout," he says, with the aim being the creation of an entire system in a handheld device, perhaps even for use in an ambulance. SNP Preparing samples for Eureka Genomics' (www.eurekagenomics.com) Mass Genotyp- ing by Sequencing Technology methodology starts with heating DNA to melt it apart and break it into smaller pieces, "which makes the hybridization of the next set of probes onto it much easier," explains John Curry, Ph.D., senior scientist. For each single nucleotide polymorphism (SNP) to be interrogated three barcoded probes are then added: a phosphorylated right hybridization sequence, and two left hybridization sequences, which differ by a complementary SNP and permit discrimina- tion between the two different alleles in the genomic sequence. SKILLED & ACCOMPLISHED CAPABILITIES All Modifications and Oligo Types Synthesized • Long oligos up to 250 mer • Fluorescent Molecular Probes • Ultra-Modified, DNA, RNA, Chimeric, Fluorescent, and Antisense Oligos • Specializing in the design and synthesis of challenging combinations of modifications Providing oligos for demanding applications and consistent results for more than a decade Gene Link. Results you can rely on. 1-800-GENE LINK www.genelink.com 190 Saw Mill River Road | Hawthorne, NY 10532 tel: 914-769-1192 | fax: 914-769-1193 Gene Link™ 30 | October 1, 2012 | genengnews.com | Genetic Engineering & Biotechnology News The hybridized probes are then ligated and act as a template for the subsequent PCR reaction, which further adds sample specific indexes. The assays (one sample, but hundreds of loci, per well) begin in 384-well plates and are combined and spun down into a small library "so we're really taking a few milliliters of PCR products and reducing it down to 100 µL of library," explains Dr. Curry. "And a portion of that library goes into the sequencer." This type of ligase discrimination for SNPs has been done for 20 years. Yet "whereas be- fore people would do this assay one at a time, or 40 at a time, and resolve it on a PAGE gel, we're resolving it on a next-gen sequencing platform," he continues. "So we're able to put thousands of sam- ples, with hundreds of loci, into a single tube, onto a single lane of an instrument, and get the information back, and decipher it, and determine the genotypes for basically 1,000 x 100 SNP-sample combinations." The assay never actually has to read the biological information itself "because they're all on the probes that are designed from the biological information," notes Dr Curry. This allows for shorter, more eco- nomical reads. In addition the barcodes can be multiplexed. "We're able to drive the cost down to frac- tions of a cent per animal SNP-combination, and do them all at once." Eureka Genomics has already commercialized this process for agricultural and clinical applications. An actual gel photo of each oligo is affixed on the oligo report. An absolute testimony of quality.