Assessment of DNA extraction methods for PCR testing of discontinued or unapproved biotech events in single seeds of canola, flax and soybean

Abstract

The purpose of the study was to assess the suitability of three DNA extraction methods (Fast ID, SDS-based and relatively high-throughput methods) for single seeds (kernels) of canola, flax and broken pieces of soybean seeds. The extracted DNA was used for conventional and real-time qualitative PCR detection of the biotech events OXY235 canola, FP967 flax and DP305423 soybean. The mean weight of single canola and flaxseeds ranged from 2.3 to 3.2 mg and 5.5 to 5.8 mg, respectively. For canola, mean total DNA yields of 216, 796 and 377 ng per seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. For flax, mean total DNA yields of 329, 826 and 957 ng per seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. For soybean, mean DNA yields of 58, 102 and 175 ng per mg of broken seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. Comparison of mean DNA yields indicated statistically significant differences among the three DNA extraction methods for all the three grain types. There was either weak or no correlation between seed weight and DNA yield for all the three DNA extraction methods. Abs260/280 ratio of ≥1.8 was obtained for DNA extracted with Fast ID and SDS-based methods. DNA isolated with all three extraction methods had low Abs260/230 ratios indicating the presence of contaminants that absorb at 230 nm. Consistent and repeatable PCR amplification was obtained for DNA extracted with the Fast ID and SDS-based extraction methods. Inhibition of PCR was observed for flax DNA extracted using relatively high-throughput method; however, reducing the amount of DNA to 10 ng in the PCR resulted in consistent amplification. Overall, all three DNA extraction methods can be used for fast DNA-based detection of unapproved or discontinued biotech events in single seeds of canola and flax as well as broken pieces of soybean seeds.