Development of a Sensitive, Easy and High-Throughput Compliant Protocol for Maize and Soybean DNA Extraction and Quantitation Using a Plant-Specific Universal Taqman Minor Groove Binder Probe

Abstract

We report the optimization of a high-throughput, compliant DNA extraction method that uses standard format 96-well plates and a commercial automated DNA purification system (ABI PRISM® 6100 Nucleic Acid PrepStation). The procedure was set up for maize and soybean, the most common GMO crops and the main ingredients of several foodstuffs, and compared with an EU-validated CTAB-based method. Optimization of the DNA extraction was achieved by applying self-prepared buffers (for DNA extraction, binding, and washing) on the PrepStation loaded with proprietary glass-fiber-coated purification plates. Quantification of extracted DNA was performed by real-time PCR using previously reported endogenous soybean lectin and maize starch synthase genes and a novel plant-specific universal TaqMan MGB probe that targets the 18S rRNA multiple copy gene. Using serial dilutions of both maize and soybean genomic DNAs, we show low PCR sensitivity and efficiency for the official TransPrep DNA extraction protocol compared to the CTAB-based one. On the other hand, using serial dilutions of a standard reference plasmid containing a 137 bp sequence cloned from the 18S rRNA plant-specific ribosomal gene, we demonstrate the high PCR sensitivity and efficiency of the optimized DNA extraction protocol setup with self-prepared buffers. The limits of detection and quantification of the 18S rDNA reiteration were consistent with the calculated values, supporting the suitability of the DNA extraction procedure for high-throughput analyses of large populations and small amounts of tissue.