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Bioscience Methods 2012, Vol.3, No.1, 1
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6
http://bm.sophiapublisher.com
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Research Article Open Access
Evaluation of Different Reaction Systems for HRMAnalysis inApple
Mudan Bai , Caihong Wang , Hao Yin , Yike Tian , Jiefa Li
College of Landscape and Horticulture, Qingdao Agricultural University, Qindao, 266109, P.R. China
Corresponding author email: chinanoahl@163.com;
Author
Bioscience Methods 2012, Vol.3, No.1 doi: 10.5376/bm.2012.03.0001
Received: 16 Nov., 2011
Accepted: 26 Dec., 2011
Published: 29 Jan., 2012
This article was first published in Molecular Plant Breeding in Chinese, and here was authorized to translate and publish the paper in English under the terms of
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Preferred citation for this article as:
Bai et al., 2012, Evaluation of Different Reaction Systems for HRMAnalysis in Apple, Bioscience Methods, Vol.3, No.1 1
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6 (doi: 10.5376/bm.2012.03.0001)
Abstract
High resolution melting (HRM) analysis is a newly developed method for fast DNA polymorphism detection. HRM
analysis under different reaction volumes, DNA concentrations or polymerase chain reaction (PCR) annealing programs were
evaluated for genotyping in apple with cultivar ‘Fuji’ and ‘Telamon’. The result indicated that 5μL reaction volume was as efficient
as 10μL or 20μL for revealed the polymorphism of SSR (simple sequence repeat) marker CH03d11, which derived from apple
genome, between the two cultivars. Therefore, even DNA concentration as small as 0.25 ng/μL was good enough for PCR
amplification and the following HRM detection under a 5μL reaction volume. Additional study demonstrated that a touchdown PCR
program could also performed very well in HRM analysis for polymorphism detection.
Keywords
Apple (
Malus domestica
); HRM; Reaction volume; DNA template concentration; Touchdown PCR
Background
High resolution melting (HRM) analysis is a new
genotyping technology based on the physical properties
of nucleicacid for resolution melting analysis of PCR
products by using of high resolution instruments with
special
“
saturation
”
dyes (Tan et al., 2009). HRM
curve analysis was performed using the HRM module
of Lightscanner32, LightCycler
®
480 II and Rotor-Gene
6500 and so on. In addition, special fluorescent dyes
in use include LC Green, Eva Green, SYTO 9 and
LightCycler 480 ResoLight Dye, which can strongly
bind to double-stranded DNA in a saturated manner
without any PCR inhibition (Wu et al., 2009; Norambuena
et al., 2009). Therefore, dsDNA is not rearranged in the
process of denaturation at high temperature, allowing
the melting curve has a higher resolution and specificity
(Chen et al., 2009).
High resolution melting analysis has several advantages
over traditional SNP and quantitative probe methods.
For example, (a) high-throughput
—
the HRM can
analyze 96 or 384 simples at the same time; (b)
high-sensitivity
—
its sensitivity can reach 1% to 0.1%,
which is 25~250 times than that of the traditional PCR
and Sanger sequencing methods; (c) good specificity
—
the
specificity of PCR products approach 100%, and these
products are without further processing, achieve
closed-tube operation and avoid cross-contamination
(Li et al., 2009); (d) simplicity
—
PCR reaction can be
carried out only with PCR primers without specific
probes and sequencing, and the sample genotype can
be judged completely by HRM curve directly; (e)
high-security
—
PCR products needn’t been validated
by gel-electrophoresis, which can avoid the technicians
using the toxic or harmful reagents and UV lamp
(Vossen et al., 2009). Therefore, HRM greatly simplifies
the procedure and reduces the time of analysis, which
has broad application prospects. Nowadays, the HRM
technique is useful for genotyping (Wittwer
et al.,
2003), varietal identification (Mackay et al., 2008),
microsatellite markers analysis (Mader et al., 2008),
sequence matching (Zhou et al., 2004), mutation
scanning (Dong et al., 2009) and DNA methylation
(White et al., 2007; Wojdacz and Dobrovic, 2007) and
other investigation fields.
LightCycler 480 II, introduced by the Roche Applied
Science, is a HRM instrument for high-throughput