GD of first derivative of high-resolution melt curves of amplicons from gDNA of unmodified target sites.

<p>gDNA from mock-transfected HEK293T cells (Mocks) were PCR amplified using primer pairs targeting F8-S2 or CCR5 loci to obtain high resolution melt curve data as described in Materials and Methods. The normalized and fluorescence corrected melt curve data (nFcRFU) from F8-S2 (A and C) and CCR5 (B and D) target sites were numerically differentiated as described in Materials and Methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190192#pone.0190192.e008" target="_blank">Eq 7</a>). 1-GD (A and B) and 2-GD curve fitting of derivative melt curves were done using CurveExpert Professional using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190192#pone.0190192.e010" target="_blank">Eq 9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190192#pone.0190192.e011" target="_blank">Eq 10</a>, respectively. The first derivative (y-axis: -d(nFcRFU)/dT) was plotted against temperature (x-axis) and is shown as blue dots. The 1-GD curve fit to the first derivative data is shown as a red trace in A and B. The individual Gaussians of 2-GD curve fit are shown as brown (g2(x)) or green dashed lines (g3(x)) and their sum (g2(x) + g3(x)) is depicted as a solid red line in C and D. Table E shows the Gaussian parameters determined from 1-GD curve fitting of A and B, while Table F shows the parameters identified by 2-GD curve fitting of C and D using the CurveExpert Professional software.