Page 8 - BM 2012 vol.3 No.1

Bioscience Methods 2012, Vol.3, No.1, 1 -6
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5
We also confirmed this phenomenon in apple.
Previous reports that the volume system of HRM
analysis is basically over 10 μL. But the scientists
found that the volume reaction system of 5 μL was
still good to polymorphism detection and genotyping,
and other reagent consumption was one-fouth of 20 μL
system recommended by the kit. The cost of HRM
analysis has been reported as $1.50 per sample in 20 μL
reaction system (Wu et al., 2008). Hence, without
affecting the test results, 5 μL reaction system can
greatly reduce costs. In addition, we also found when
the DNA template concentration was 0.25 ng/μL, the
HRM detection of the PCR amplicon can be carried
out normally. Therefore, when the amount of DNA
template is a little, we can consider a lower concentration
in order to make sure the multiple experiments.
Touchdown PCR is a common analysis model in PCR
amplification, its annealing temperature gradually
dropped from a high value to a lower value with the
amplification cycle. Then the following expansion
cycle will be completed with this low value. This
model can be normally amplified and reduce non-specific
amplification.
The study showed that the touchdown PCR could get
the same typing with the conventional PCR in the
different reaction volume. Because touchdown PCR
provided correct annealing temperatures for many
primers at the same time, which would make it
possible to amplify and analyze the multiple markers
in the same run. It is important to improve the
efficient use of the materials and the equipments.
3 Materials and Methods
3.1 Plant materials
Apple cultivars ‘Fuji’ and ‘Telamon’ were used in the
study.
3.2 Genomic DNA extraction
DNA was extracted from fresh young buds (about
0.1 g) using modified CTAB method (Tian et al.,
2003). DNA was quantified on a Ultrospec 3300 pro
(Amersham Biosciences) and conserved at -20℃
until we needed.
3.3 PCR amplification and HRM verification
We used the SSR markers CH03d11 linkaged to apple
columnar trait as the tested object. PCR amplification
and HRM analysis were performed on the LightCycler®
480 II Real-Time PCR System in 96-well multi-well
plates, and PCR reactions consisted of 1×LightCycler®
480 High Resolution Melting Master; supplemented
with 2.0 mmol/L MgCl2 and 0.2 μmol/L each primer.
We totally arrange three kinds of treatments for the
reaction volume: 5 μL, 10 μL and 20 μL and four kinds
of treatments for DNA template: 2 ng/μL, 1 ng/μL,
0.5 ng/μL and 0.25 ng/μL. The cycling program
consisted of a universal PCR protocol as follows:
pre-denaturalization for 10 minutes at 95℃ , 95℃
denaturalization for 10 seconds, 60℃ annealing for 15
seconds and 72 ℃ extension for 10 seconds. The
amplification cycles were immediately followed by
the high resolution melting steps of 95℃ for 1 minute,
cooling to 40℃ for 1 minute, raising the temperature
to 65℃ and then raising the temperature to 95℃ with
25 fluorescent acquisitions per degree Celsius in this
step. In addition, a touchdown protocol has a similar
amplification cycles but with annealing temperatures
decreasing from 60 ℃ to 55℃ for 15 seconds. The
annealing temperature decreased in subsequent cycles
by 0.5℃ per cycle after the first 60℃ annealing step
to 55 ℃ (Yin et al., 2011).
3.4 High resolution melting analysis
The melting curve was analyzed with the gene
scanning software module (1.5 version) on the
LightCycler® 480 II instrument.
Authors' contributions
CHW and MDB conceived the experimental design and
objectives of all the HRM experiments, conducted the HRM
data analyses, and wrote the manuscript. HY and JFL
conducted a few data analyses and took an active part in
experimental design method. CHW and YKT were the prime
principal of the project and took part in reviewing and writing
the manuscript. All authors have read and approved the
manuscript.
Acknowledgements
This work was co-supported by Shandong province thoroughbred
Project and Qingdao Municipal Science and Technology
Program of Basic Research Projects (11- 2-4 -5 -(4)-jch).