The purifi ed DNA from 25 samples had a median concentration of 19.1 ng/L and ranged from 1.2 to 53.4 ng/L. Consequently, a number of the genotyping reactions used more than the 20 ng of DNA template recommended by the manufacturer.
Allelic discrimination plots
Figure 1 shows the allelic discrimination plot for the thymidylate synthetase assay and Figure 2 shows the plot for the apolipoprotein E assay. The plots are presented as the signal (average fluorescence between cycles 41 and 45) minus the background (average fluorescence between cycles 21 and 25).
Discussion and Conclusions
TaqMan assays are widely used for SNP genotyping. As with other molecular genetic techniques, the quality and purity of DNA is important for reliable results. Our findings indicate that DNA samples collected and purified using Oragene kits are suitable for allelic discrimination using TaqMan probes. Clear discrimination between different genotypes is evident in Figures 1 and 2.
Although it is recommended to use 1 to 20 ng of DNA per reaction, this
study used DNA amounts ranging from 1.2 to 53.4 ng and all 25 samples
gave clear, interpretable results. It was possible to perform the reactions
without first determining the amount of DNA. Thus, the step of adjusting
the DNA concentrations co