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SELECTBIO Conferences PCR & Next-Gen Sequencing


Novel Methods of Detecting Mutations and Copy Number Variation with Single Color Digital PCR

Hanlee Ji, Assistant Professor/Senior Associate Director, Stanford University School of Medicine

Droplet digital PCR (ddPCR) is a robust technology for quantitating genetic variation with high sensitivity from minimal amounts of nucleic acid. Compared to the highly variable quantitative real-time PCR (qRT-PCR), ddPCR eliminates relative standards and has the advantage of measuring mutant and wild-type targets within the same well. Commonly, this is achieved with the use of a two-color fluorescent oligonucleotide probe (TaqMan) design, where the mutant is represented by a FAM probe and the wild type by a VIC or HEX probe. In this study we present an alternate quantification method for assessing CNVs and SNVs using a non-specific DNA binding dye. Instead of two colored probes, our design manipulates the length of the region of interest (ROI) and control amplicons to distinguish between their fluorescent signals. The dye binds in greater amount to the longer length target giving a higher fluorescent signal than the mutant target and both populations are easily quantifiable. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay. The ability to customize this assay for a variety of functions is a major advantage of using a single-color dye. Here, we demonstrated the effectiveness of this method by assessing copy number of the proto-oncogene FLT3, a target for the small molecule inhibitor Sunitinib. We established accurate quantitation of a FLT3 copy number change in a tumor sample harboring a 1.5-fold amplification diluted to 20% in a normal sample. Additionally we explored the common V600E point mutation in BRAF and an activating mutation in HER2, a potential therapeutic target for cancers lacking HER2 amplification. Our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample and in the case of HER2, distinguish between wild-type and two different mutant alleles. Aside from single base pair mutations and copy number analysis we used t

Add to Calendar ▼2014-04-29 00:00:002014-04-30 00:00:00Europe/LondonPCR and Next-Gen