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Bioscience Methods 2012, Vol.3, No.1, 1
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analysis. But the price of the relative reagents is high. In
this paper, we compared the analysis effects on different
HRM systems by
Malus domestica
to explore an
effective way that can reduce the cost of the HRM
analysis. And it would be very important for further
expanding the application of this technique in fruit trees.
1 Results and Analysis
1.1 Effect evaluation of different reaction volume
for HRM analysis
In this experiment, HRM analysis showed that SSR
marker CH03d11 between ‘Fuji’ and ‘Telamon’ can
arise similar DNA polymorphism melting curve in
three kinds of different total reaction volume (Figure 1).
This polymorphism changes can be shown by the
view of three different forms: (1) the normalized and
shifted melting curve (Figures 1A), which is adjusted
all the samples in the context of the same temperature
to compare the relative fluorescence signal values; (2)
the normalized and temperature-shifted difference plot
(Figures 1B), which selects one sample as the melting
curves plotted baseline to highlight the relative
differences in samples; (3) the melting peaks (Figures
1C), a derivative
type, which is to compare the change
rate of fluorescence signal between the samples with
temperature, namely the fluorescence value and
temperature. In addition, the peak corresponding to
the specific temperature is the melting temperature
(Tm) of the samples. The differences between samples
can be obviously seen from the curve of the first graph,
however, the second graph can definitely distinguish
the differences in the samples, and we can easier to
identify the samples. From the third graph, we can
intuitively understand the difference in different
samples’ melting temperature.
Figure 1 Effect evaluation of different reaction volume for HRM analysis
Note: A: Normalized and shifted melting curve; B: Normalized and temp-shifted difference plot derived from A; C: Melting peaks
derived from A