Single-color Multiplexed Digital PCR via High Resolution Melt Analysis of Endpoint Digital PCR Partitions

AbstractDigital PCRdPCRtechnology has drawn attention in both the genomic research and clinical research communities for its ability to detect rare eventshigh sensitivitybe less prone to inhibitionhigh specificityquantify without a standard curvehigh precisionand exploit well developed qPCR reagentsHowevercurrent dPCR platforms are slow to overtake the current qPCR gold standard due to their high consumable costsexcessively complicated workflows and incompatibility with automationCOMBiNATiandapos s integrated single instrument and micromolded consumable technology will deliver the first turnkey dPCR platform with efficient running cost for absolute nucleic acid quantificationIn this studywe will exploit our integrated instrumentation to be the first ever to demonstrate single color dPCR multiplexibility using High Resolution MeltHRManalysis of end point dPCR partitionsFor this PhaseSBIR projectthe collaboration of COMBiNATi co founder DrMegan Dueck and clinical HIV specialist DrSteve Yukl will strive to prove the feasibility of a novel multiplexedsingle color HRM dPCR assay for studies involving the latent HIV reservoirUnderstanding the mechanisms leading up to HIV latency is paramount to finding an ultimate HIV cureDuring themonth PhaseSBIR projectCOMBiNATi co founder DrMegan Dueck in collaboration with DrSteve Yukl will complete the following key deliverablesDesign and prototype a large field of viewhigh sensitivity optical module for realtime imaging of individual dPCR reaction partitions during incremental increase intemperatureAs the first single instrument dPCR platformCOMBiNATi strives to bethe first to show multiplexedsingle color nucleic acid quantification using HRM analysisof endpoint dPCR partitionsToward this end we will build an optical module capable ofgenerating target specific HRM curves for individual end point dPCR partitionsDevelop a single colormultiplexed HRM dPCR assay fortranscription profilingof the latent HIV reservoirOur novel HRM dPCR multiplexing strategy will bebenchmarked using a panel ofpre validated dPCR assays that quantify markers fordifferent stages of HIV transcriptional blockadeWe hope to ultimately develop a robustsingle color dPCR assay for examining mechanisms of HIV latency Narrative Despite its promisedigital PCRdPCRis slow to overtake the traditional quantitative PCRqPCRgold standard as thego totechnology for targeted nucleic acid quantificationCOMBiNATi strives to accelerate this adoption with our fully integratedstreamlined and costeffective dPCR platformIn this studywe aim to be the first ever to achieve single color dPCR multiplexibility using High Resolution MeltHRManalysis of end point dPCR partitionsTo benchmark this novel multiplexing strategy we will develop a method for measuring multiple markers for HIV transcription blockade within the same reactionThe assay we develop will be a valuable tool for studies involving the latent HIV reservoir