ASSESSMENT OF DNA EXTRACTION METHODS FOR GMO ANALYSIS FOR GRAIN MONITORING IN MEXICO. PART II: QUANTIFICATION BY REAL-TIME PCR

Because of their specificity and high throughput, real-time PCR-based methods are suitable for the monitoring of genetically modified (GM) organisms at field level or in the grain trade, in order to comply with federal regulations on biosafety. In the first part of this study, DNA extracted from different maize (Zea mays L.) tissues using several commercial purification protocols available in Mexico were evaluated in terms of DNA quality as substrates for end-point PCR. In this second part, DNA preparations obtained from grain, by means of the same commercial protocols, were tested in quantitative PCR (qPCR), using TaqMan and SYBR Green protocols. Linear dynamic range, amplification efficiency and method accuracy were assessed using recommended qPCR criteria for validation purposes. Results showed that the chemical complexity of plant tissues, such as grains, require an efficient purification protocol for consistent and reliable PCR quantification. The ratio A(260/280) and DNA visualization in agarose gels are recommended as preliminary-but not exclusive- criteria of DNA quality because DNA amplification capability is not evinced with these procedures. Two of the methods tested yielded good-quality DNA as revealed by the linear dynamic range analysis. The third method analyzed rendered inaccurate results due to the presence of grain endogenous PCR inhibitors that did not allow a proper DNA quantification. As conclusion, TaqMan chemistry showed to be more sensitive to the presence of impurities than SYBR Green, even though amplification could be achieved in the latter, quantification may not be accurate. Silica DNA-binding membranes yield the most suitable DNA preparations for PCR quantification of GM maize grains.