Table 1.
Primers and probes used in this study.
Figure 1.
Preparation of plasmid samples.
Lane M: 1 kb ladder DNA marker. Lane 1: S, supercoiled plasmid sample. Lane 2: L, linear plasmid sample (SspI treated). Lane 3: N, nicked-circular plasmid sample (Nt.BspQI treated). Lane 4: C, closed-circular plasmid sample (topoisomerase I treated). Lane 5: S-LB, supercoiled plasmid treated with SspI reaction buffer. Lane 6: S-NB, supercoiled plasmid treated with Nt.BspQI reaction buffer. Lane 7: S-CB, supercoiled plasmid treated with topoisomerase I reaction buffer.
Figure 2.
Effect of plasmid DNA conformation on DNA measurement methods.
(A) Comparison of various methods on supercoiled plasmid DNA quantification. (B) Hoechst dye-based DNA quantification method. (C) Quant-iT dsDNA BR quantification assay. (D) OD260 DNA quantification method. S-LB: supercoiled plasmid treated with SspI reaction buffer. S-NB: supercoiled plasmid treated with Nt.BspQI reaction buffer. S-CB: supercoiled plasmid treated with topoisomerase I reaction buffer. a, b, c, d and e : groups with significant difference to each other (Duncan's multiple range test, P<0.05).
Figure 3.
Effect of plasmid DNA conformation on qPCR calibration curves.
Each data point was the average of three triplicate tests (n = 9). Blue lines: Supercoiled plasmid calibration curves. Red lines: Linear plasmid calibration curves. Gold lines: Nicked-circular plasmid calibration curves. Magenta lines: close-circular calibration curves. Grey lines: Buffer control (S-LB, S-NB and S-CB, supercoiled) plasmid calibration curves.
Table 2.
The PCR efficiency of four real-time qPCR systems on various plasmid DNA conformations.
Figure 4.
Effect of plasmid DNA conformation on qPCR chemistries.
Relative amplification was calculated by the ΔCt value to the corresponding supercoiled plasmid group. Each data point was the average of three triplicate tests (n = 9) with 200 copies of plasmid DNA sample. * : P<0.05. *** : P<0.001.
Table 3.
SYBR Green I qPCR quantification of GM maize NK603 based on supercoiled and linear plasmid DNA calibration curves.
Table 4.
Comparison of 0.5% NK603 CRM quantifications based on suprcoiled and linear plasmid DNA calibration curves.
Figure 5.
Three hypothetical type of DNA amplification in qPCR.
Type I: The amplification from DNA template (plasmid). Type II : The amplification from PCR product of type I. Type III : The amplification of PCR product itself.
Figure 6.
The proposed and virtual effect of initial PCR efficiency (Ei) on qPCR amplification curves (assuming Eii = Eiii).
Ei (initial PCR efficiency): the efficiency of type I amplification. Eii: the efficiency of type II amplification. Eiii: the efficiency of type III amplification. (A) Proposed amplification curves with 100% (theoretical), 10% and 1% Ei. (B) Proposed amplification curves of 100% and 20% Ei with 10 and 100 copies of DNA template (initial copy number, Ci). (C) Proposed amplification curves of 100% and 20% Ei with 10 and 100 Ci in PCR. Difference in Ei will consistently shift Ct value (by the change of Y-intercept). (D) PCR amplification curves of supercoiled and nicked-circular plasmid DNA with 2,000 and 20,000 Ci in this study.
Table 5.
The effect of initial PCR efficiency (Ei = 100% and 20%) on PCR product yield.