Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events

Abstract

Low-level detection and quantification of genetically engineered (GE) traits with polymerase chain reaction (PCR) is challenging. For unapproved GE events, any level of detection is not acceptable in some countries because of zero tolerance. Droplet digital PCR (ddPCR) has been successfully used for absolute quantification of GE events. In this study, reliability of low level quantification of GE events with ddPCR was assessed using a total of 50, 100, 200, 400, and 600 ng DNA spiked at 0.01% and 0.1% concentration levels. Genetically engineered canola (GT73 and MON88302 events) and soybean (A2704-12 and DP305423 events) events were used for the study. For samples spiked at 0.1% level, reliable quantification was achieved for the four GE events using 50 or 100 ng DNA. Few target droplets were generated for 0.01% spiked GE samples using 50 and 100 ng DNA. Increasing the amount of DNA for ddPCR generated more number of target droplets. For GE canola events, the use of 400 and 600 ng DNA for ddPCR resulted in saturation. The use of multiple wells of 200 ng DNA (instead of 400 and 600 ng per well) helped to overcome the saturation problem. Overall, the use of high amount of DNA for ddPCR was helpful for the detection and quantification of 0.01% GE samples.