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

0.993 was obtained. Two samples of human genomic DNA were also tested. By interpolation of the standard curve, the first patient sample was determined to have an initial concentration of 254 copies/l, the second had 357 copies/l.

Specificity of the PCR was measured in two ways. First, melting curve analysis was performed on each sample. For example, the melting curve analysis for patient sample 1 revealed a single peak at 88.5C, which indicates exclusive amplification of the -actin amplicon as seen in Figure 4. Second, gel electrophoresis was performed to analyze the PCR products. Figure 5 reveals single bands for each standard and sample, indicating specific amplification of the desired product with only minor amplification of a primer-dimer.


Discussion
Broad Dynamic Range for Lambda and Human DNA
DyNAzyme II DNA polymerase can be used successfully with SYBR Green I dye to quantify DNA from small genomes as well as the more complex human genome. Experiments revealed a linear relationship between log starting copy number and C(t) value over at least 6 orders of magnitude for the lambda genomic DNA template, and over at least 3 orders of magnitude for the human genomic DNA template. The broad dynamic range of starting template that can be detected makes this assay particularly valuable when sample DNA concentrations are unknown or vary widely.

In this study, the lower limit of the standard range was 100 copies of lambda DNA, or 75 copies of human genomic DNA per reaction. Other studies have shown linear results below this level. Though not necessary in this application, detection down to several copies of template could b
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