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1.3 Effect evaluation of touchdown PCR protocol
for HRM analysis
In the HRM reaction, it is very critical to select an
appropriate annealing temperature (Mackay et al.,
2008; Erali and Wittwer, 2010). HRM analysis effects
were compared between the touchdown PCR protocol
and the conventional PCR protocol. The result showed
that these two kinds of amplified modes can get the
same test results when the reaction volume was 20 μL,
10 μL and 5 μL, repectively (Figures 3).
Figure 3 Effect evaluation of touchdown PCR protocol 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
2 Discussion
Currently, HRM technique has been widely adopted in
human disease-related gene mutation scanning and
molecular diagnostics fields, and formed a relatively
complete research system (Luo et al., 2011). Recently,
there are a few reports on HRM in plants. Such as, the
identification of the different varieties of grapes and
olives (Mackay et al., 2008); SNP marker analyses of
expressed sequence tags from apple (Chagné et al.,
2008);
the identification of barley gene locus
(Hofinger et al., 2009) and so on. Recently, Li et al
(2011) have explored that the reaction system of HRM
using rice as the test materials and the results showed
that the stability of the volume of 5 μL reaction was
worse than that of 10 μL, 20 μL reaction systems and
caused some deviations of the amplicon’s Tm value.