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Bioscience Methods 2012, Vol.3, No.1, 1
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Seeing from Figure 1, the three different view forms
can be clearly reflected the polymorphisms between
the two cultivars, and the reproducibility of the
experimental results is good in the different total
reaction volume. As shown in Figure 1C, the change
of reaction volume caused some deviations of the
amplicon’s Tm value, fortunately, this bias did not
affect the overall analysis effects.
1.2 Effect evaluation of different concentration of
DNA for HRM analysis
The concentration of DNA template is one of the main
factors to affect PCR amplification. Generally, PCR
amplification can normally carry out as DNA template
concentration in a large range. It will result in no
amplification product or produce non-specific
amplification, while the template concentration is too
high. When the template concentration is too low, it is
also not conducive for effective PCR amplification.
All these will directly affect the subsequent HRM
signal detection. In this study, we set a gradient
experiment for the DNA template concentration with
0.25 ng/μL, 0.5 ng/μL, 1.0 ng/μL, and 2.0 ng/μL in the
total reaction of 5 μL. The result showed that the
analysis of the samples is similar in the different DNA
template concentration (Figures 2).
That is to say,
when the lowest concentration of DNA template is
one-eighth of the highest, HRM can detect the PCR
amplicon normally.
Figure 2 Effect evaluation of different concentration of DNA 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