RNASE A enzyme modification of optimized SDS protocol for DNA extraction suitable for real-time PCR screening of GMOs

Abstract

As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding RNase A enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 μl of RNase A enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 μg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/μl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.

Keywords

• 5s
• tnos
• dna purity
• yield
• real-time pcr