Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

Publication date: September 2014 Source:Biomolecular Detection and Quantification, Volume 1, Issue 1 Author(s): Coren A. Milbury , Qun Zhong , Jesse Lin , Miguel Williams , Jeff Olson , Darren R. Link , Brian Hutchison Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 w...
Source: Biomolecular Detection and Quantification - Category: Molecular Biology Source Type: research