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OMICS Further denaturation of these duplexes at critical denaturation temperatures preferentially releases the mismatched strands, which then can be amplifed by PCR. Two consecutive rounds of COLD-PCR result in a 100-fold enrichment of mutations. "Further enrichment with COLD-PCR was challenging due to polymerase errors and mis-priming events," Dr. Makrigiorgos says. "We used the same scientifc principle to develop an alternative enrichment method that does not involve enzymatic amplifcation." DISSECT (differential strand separation at critical temperature) also uses differential denaturation of DNA heteroduplexes. However, it avoids enzymatic steps being entirely based on repeated cycles of denaturation and hybridization on magnetic beads coated with wildtype target gene sequences. Wild-type DNA remains attached to the beads, while the mutant DNA is released and collected. Because the sequences are not altered during DISSECT, the method is compatible with downstream applications including qPCR and sequencing. "The method is exceedingly simple, and is easy to automate, multiplex, and scale up," explains Dr. Makrigiorgos. "DISSECT can simultaneously enrich diverse targets for multiple mutations in the same tube using a single denaturation temperature." In a proof-of-principle study, three to four rounds of DISSECT produced up to 100- to 400-fold enrichment of mutations in three selected oncogenes" based applications do, Lights-On/Lights-Off uses the same color to detect many mutations at once in the same tube. Each Lights-On probe consists of a quencher and a fuorescent moiety, and Lights-Off probe has only a quencher moiety. Each Lights-Off probe quenches the Lights-On probe when both are bound to the target in a close proximity. The signals from all contiguous Lights-On probes create a composite fuorescent contour, which is mathematically converted into a sequence-specifc fuorescent signature. The fuorescent probes will bind to mismatched sequence at a lower temperature and will produce a distinct fuorescent contour. "We found that each mutation in lactamase's hot spots produced its own specifc signature," Dr. Pierce says. Brandeis University owns LATE-PCR and its allied technologies and is prepared to license them for commercialization. "Current diagnostic tests rarely provide immediately actionable information," asserts David Dolinger, Ph.D., evp, Seegene. "Medicine, and in particular diagnostic medicine, needs to evolve from art to science where diagnostic assays are based on signs and symptoms of disease." Seegene believes two of its technical achievements will transform diagnostics. Dual Priming Oligonucleotides (DPO™) eliminate typical "noise" problems in multiplex PCR. DPO primers consist of two distinct annealing regions separated by a unique polydeoxyinosine linker. The larger portion, called the "stabilizer," binds to target DNA resulting in stable annealing. However, extension will occur only if the shorter arm selectively binds to the target sequence. Built-in thermodynamic constraints create an internal control so that specifc extension only occurs when both arms anneal. "The DPO technology opened doors for literally unlimited multiplexing," Dr. Dolinger affrms. "The second piece of the puzzle required solving the detection of the targets in a multiplex reaction. Current fuorescent detection technologies can only deliver three to fve answers per sample. We wanted to achieve at least an order of magnitude more." Seegene's approach involves detection and measurement of the "catcher," an artifcial single-stranded template labeled with a quencher and a fuorescent moiety. In its free form, the catcher does not fuoresce. And then there is the "pitcher," a singlestranded nucleotide composed of a sequence that binds to a target sequence that also has a tagging portion. As the PCR reaction initiates, the DPO primers and the pitcher hybridize to the target gene. Once the DNA extension reaches the pitcher, Taq polymerase with 5' nuclease activity cleaves the tagging portion. The tagging portion uniquely binds to the catcher and, if cleaved at the correct base, could be extended to form the duplex catcher. The duplex causes the physical separation of the quencher and the fuorophore, producing a fuorescent signal. Keeping a watchful eye on cytosolic DNA. Finding Multiple Mutations "Many important diagnostic applications require discrimination of multiple sequence variants present in the same sample," says Kenneth Pierce, Ph.D., senior research scientist at Brandeis University. "Under these conditions, technical challenges for detection based on the classical probe-target hybridization are signifcant. We developed a novel PCR-based approach that overcomes these challenges and opens doors for a variety of diagnostic and species identifcation applications." This approach, linear-after-the-exponential (LATE)-PCR is an elegant adaptation of the asymmetric PCR method. Because of the unique primer design, LATE-PCR effciently generates single-stranded DNA after the period of exponential double-stranded amplifcation. Single-stranded DNA is a superior target for product detection using complementary DNA probes. "Single-stranded DNA offers an opportunity to use low annealing temperatures for detection," Dr. Pierce says. "This means that a single universal probe can be used to detect sequences of high diversity. Conversely, we can use the temperature gradient to obtain information about specifc mutations in a given sequence." The team used this novel approach to study antibiotic resistance of gram negative bacteria. Mutations in beta lactamase enzymes give rise to an extended spectrum of antibiotic resistance. Rapid detection of a specifc mutation may help with selection of the appropriate medical treatment. One detection approach combines LATEPCR with Lights-On/Lights Off probes. Rather than detecting each mutation with its own color probe, as many current PCR- Cytosolic dsDNA Sensors Innate immune detection of intracellular DNA derived from viruses and invasive bacteria is very important to initiate an effective protective response. This crucial step depends on cytosolic DNA sensors (CDSs), which, upon activation, trigger the production of type I interferons (IFNs) and the induction of IFN-responsive genes and proinfammatory chemokines. InvivoGen has developed innovative tools in order to facilitate the study of CDS. An expanding range of tools CDS Ligands CDS Reporter Cells CDS Inhibitors Functionally tested ligands Sterile VacciGradeTM for use in vivo www.invivogen.com/cds INNOVATION WITHIN REACH Genetic Engineering & Biotechnology News | GENengnews.com | August 2013 | 25