Concordance Study: Methods of Quantifying Corn and Soybean Genomic DNA Intended for Real-Time Polymerase Chain Reaction Applications

Abstract

Quantitative real-time polymerase chain reaction (qPCR) is a technology commonly used for the detection and quantification of genetically engineered (GE) traits in grains and oilseeds. The method involves measuring copy numbers of taxon-specific, endogenous control genes exposed to the same manipulations as trait-specific target genes. Accurate DNA quantification is essential for successful and predictable results with qPCR. A systematic study of seven different DNA quantification methods, incorporating different chemistries and different instrumentation, were evaluated on corn and soy DNA that was extracted using two distinct extraction methods. A time course study showed that corn and soy DNA was stable under typical laboratory storage conditions. CornCTAB and cornQiagen DNA extracts produced statistically similar quantification values when measured by picogreen PG(TD700), PG(Lum20/20), Hoescht(TD700), and Hoescht(Lum20/20) methods, suggesting that these methods can be used interchangeably to quantify DNA in corn samples prior to initiation of qPCR. Soy(Qiagen) provided greater stochastic measurement variability when quantification methods were compared, whereas soyCTAB had statistically significant differences when a PG method was compared to a Hoescht method of DNA quantification. Finally, agarose gel electrophoresis data revealed more pronounced degradation for Qiagen-extracted DNA compared with CTAB extracts in both corn and soy. Consequently, Ct values generated by qPCR suggested that absolute copy numbers of PCR amplifiable targets were not concordant between Qiagen and CTAB DNA extracts. Understanding measurement uncertainty from component steps used in qPCR can contribute toward harmonizing methods for the detection of GE traits in grains and oilseeds.