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Real-Time Quantification of Genomic DNA Using DyNAzyme II DNA Polymerase and SYBR Green I Dye

emplate more quickly generate the amount of product necessary to be detected with SGI. The linear range extends from 108 to 102 copies of lambda DNA initially present in the reaction. A plot of log quantity vs. C(t) cycle for the 100bp amplicon is shown in Figure 1b. The regression coefficient is 0.991, indicating a strong linear correlation.

The precision of this assay was measured by calculating the variation in C(t) values across the four replicates at each template concentration. The standard deviation for C(t) values was found to be 0.3 units or less as shown in Table 3.

Figure 2 shows the melting curves for the four replicate lambda DNA standards containing 104 copies per reaction. The curve shows the typical smooth decline in fluorescence with an increase in temperature as the strands of dsDNA dissociate and bound SGI is released. The negative first derivative calculation is also shown. Here, the point of inflection for the curve, the melting point (Tm), is clearly seen as a single, common peak at approximately 82C. This peak corresponds to the predicted melting temperature of the amplicon, indicating amplification of the amplicon of interest. Anomalies due to contamination, primer-dimer, false priming, etc., are not evident in the negative first derivative plot, as indicated by the lack of additional peaks or of abnormal broadening of the single peak.

Human Genomic DNA Template: Patient Samples Quantified Using a Standard Curve
A standard curve generated from human genomic DNA is shown in Figure 3. A linear curve in the range of 7.5x104 copies/reaction to 75 copies/reaction with r2=
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