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

enomic DNA generate a 294bp amplicon. Sequences were: forward, 5-TCA-CCCACA- CTG-TGC-CCA-TCT-ACG-A-3, and reverse, 5-CAG-CGGAAC- CGC-TCA-TTG-CCA-ATG-G-3

Following reaction assembly, the plates or tubes were transferred to the DNA Engine Opticon real-time system, where cycling was performed according to the program listed in Table 2.

The cycle threshold C(t) line was set using the signal/noise ratio option in the Opticon Monitor software (MJ Research) set to 10 standard deviations above the mean fluorescence values for the first 37 cycles. This threshold is automatically applied to all wells and allows the comparison of standards and samples at a point that provides the most consistent results. It is critical that within any experiment, C(t) values for both standards and samples be determined using the same threshold level.

A concentration series of lambda DNA was prepared from a 0.5g/l stock. 1l of each dilution in 20l reactions was used to generate the standard curve. The standard curve for the human genomic sequence was prepared using genomic DNA from human placenta. Serial dilutions were made and analyzed to generate the standard curve.


Results
Lambda DNA Template: Linear Range and Reproducibility
Plotting fluorescence signal vs. cycle number (Figure 1a) indicates that the concentration of dsDNA in each sample rose above background fluorescence levels, and then entered a stage of exponential amplification. The C(t) values, the cycle at which the measured fluorescence intersects the cycle threshold line, decrease proportionally with the increase in initial DNA concentration. This trend is expected since higher amounts of initial t
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