質體 DNA 構形對即時定量聚合酶鏈反應定量偏差之探討

Abstract

Real-time quantitative PCR (qPCR) is the “gold standard” method of accurate quantification of genetically modified organisms (GMO). Precise quantification of target gene could be achieved by absolute quantification method based on a standard curve constructed by plasmid DNA. A recent study showed that supercoiled plasmid may suppress PCR amplification in real-time qPCR assay. In this study, we investigate the bias of DNA quantification caused by plasmid DNA conformations (supercoiled, linear, nicked circular and closed circular) both in DNA quantification methods and qPCR. Three DNA quantitative methods we used are UV absorbance at 260 nm, Hoechst 33258 method, Quant-iT assay and HPLC method. The DNA amount measured by UV absorbance is significantly higher than other methods. The effect of plasmid DNA conformations is most significantly in Quant-iT assay, and the differences among plasmid DNA conformations were between 1.62 to 1.88 fold. The qPCR systems we tested were SYBR Green I, TaqMan, TaqMan MGB and LNA TaqMan. The results showed that the standard curves which constructed with linear or nicked circular plasmid have higher relative amplifications which approximately 1.9 to 5.4 fold. The difference observed in this study may due to the difference of amplification efficiency caused by plasmid DNA conformations in the early stage of PCR. We constructed a theoretical model to elaborate possible mechanism underneath this phenomenon. The difference of PCR efficiency between supercoiled and nicked circular plasmids could be nearly 80%. In a worst-case scenario, the bias caused by DNA quantification and qPCR will be 10.2-fold. The bias of quantification which caused by plasmid conformations in the real-time qPCR assay of GM NK603 maize will neutralized due to the same bias tendency of target and reference gene standard curves. However, the plasmid conformations still have significant effect on the limit of detection of real-time qPCR assay.