Abstract
Digital PCR (dPCR) technology has drawn attention in both the genomic research and
clinical research communities for its ability to detect rare events (high sensitivity), be less prone
to inhibition (high specificity), quantify without a standard curve (high precision), and exploit
well-developed qPCR reagents. However, current dPCR platforms are slow to overtake the
current qPCR gold standard due to their high consumable costs, excessively complicated
workflows and incompatibility with automation. COMBiNATi's integrated single-instrument and
micromolded consumable technology will deliver the first turnkey dPCR platform with efficient
running cost for absolute nucleic acid quantification. In this study, we will exploit our
integrated instrumentation to be the first ever to demonstrate single-color dPCR
multiplexibility using High Resolution Melt (HRM) analysis of end-point dPCR partitions.
For this Phase 1 SBIR project, the collaboration of COMBiNATi co-founder Dr. Megan
Dueck and clinical HIV specialist Dr. Steve Yukl will strive to prove the feasibility of a novel
multiplexed, single-color HRM dPCR assay for studies involving the latent HIV reservoir.
Understanding the mechanisms leading up to HIV latency is paramount to finding an ultimate
HIV cure.
During the 9 month Phase 1 SBIR project, COMBiNATi co-founder Dr. Megan Dueck in
collaboration with Dr. Steve Yukl will complete the following key deliverables:
1. Design and prototype a large field of view, high sensitivity optical module for real-
time imaging of individual dPCR reaction partitions during incremental increase in
temperature. As the first single-instrument dPCR platform, COMBiNATi strives to be
the first to show multiplexed, single-color nucleic acid quantification using HRM analysis
of endpoint dPCR partitions. Toward this end we will build an optical module capable of
generating target-specific HRM curves for individual end-point dPCR partitions.
2. Develop a single-color, multiplexed HRM dPCR assay for “transcription profiling”
of the latent HIV reservoir. Our novel HRM dPCR multiplexing strategy will be
benchmarked using a panel of 3 pre-validated dPCR assays that quantify markers for
different stages of HIV transcriptional blockade. We hope to ultimately develop a robust
single-color dPCR assay for examining mechanisms of HIV latency.
