Significant expansion of spectral multiplexing in PCR

Project Summary There have been long-standing discussions about the role of NGS (next-generation sequencing) versus real-time PCR in clinical diagnosis, prognosis, and screening. NGS offers unrivaled discovery power and represents the ultimate in multiplexing capability. NGS, however, is expensive and often requires complex workflows with a long turnaround time. Real-time PCR, in contrast, is comparatively inexpensive and can be fully automated for operation at the point-of- care with a fast sample-in-result-out time. But real-time PCR can only detect known sequences and has a very limited multiplexing capability (typically 4-6 colors). In a clinical diagnostic, prognostic, and even screening setting, it is often sufficient to query a known panel of genes or mutations. When limited multiplexing is required, such as 4 or 5 colors in a single reaction, which can be the case in companion diagnostics, real-time PCR has been very successfully deployed. But at present, it is more difficult for real-time PCR to penetrate diagnostic or screening assays that require panels with sizes in the tens or hundreds range. For the larger panels, NGS is often used despite its more complex workflow, higher cost, and longer turnaround time. To address the above described unmet need in clinical diagnostics, this project aims to develop a new approach to achieve highly multiplexed PCR assays, for both real-time PCR and digital PCR. Given the important role PCR plays in molecular diagnostics, when successfully completed, this project is expected to greatly expand the utility and accessibility of highly multiplexed PCR assays in disease diagnosis and screening.