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

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...

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Veröffentlicht in:Food analytical methods 2021-02, Vol.14 (2), p.372-379
Hauptverfasser: Demeke, Tigst, Eng, Monika, Holigroski, Michelle, Lee, Sung-Jong
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Eng, Monika
Holigroski, Michelle
Lee, Sung-Jong
description 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.
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subjects Analytical Chemistry
Canola
Chemistry
Chemistry and Materials Science
Chemistry/Food Science
Deoxyribonucleic acid
DNA
Droplets
Food Science
Genetic engineering
Low level
Microbiology
Polymerase chain reaction
Reliability analysis
Saturation
Soybeans
title Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events
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