Direct quantification of EGFR variant allele frequency in cell-free DNA using a microfluidic-free digital droplet PCR...

Analysis of liquid biopsy samples is a promising diagnostic intervention for noninvasive detection and monitoring of cancer genotypes. However, current methods used to assess mutation status are either costly, in the case of next-generation sequencing-based assays, or lacking in sensitivity, in the case of bulk quantitative PCR measurements. Digital droplet PCR (ddPCR) is at once a sensitive and low-cost method for detecting rare cancer mutations and measuring their variant allele frequency. In this chapter, we describe a method for conducting ddPCR assays without microfluidics in a process called "particle-templated emulsification" (PTE). Using hydrogel particles and a standard benchtop vortexer to rapidly emulsify large volumes, the method forgoes the specialized instrumentation required for conventional ddPCR assays and is capable of high experimental throughput. To assess the quantitative performance of the method, we apply PTE ddPCR to analysis of variant allele frequency in EGFR, a commonly mutated gene in lung adenocarcinomas.