• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10647-10652
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• 2015

Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

• Parasites, Hosts and Diseases
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• v.51 no.6
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• pp.651-656
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• 2013

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were $84.5{\pm}0.07^{\circ}C$ and $85.7{\pm}0.07^{\circ}C$, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

• Journal of Life Science
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• v.21 no.3
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• pp.468-472
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• 2011

DNA marker is a powerful tool for plant genetics and breeding. In this study, 995 SSR markers were employed with chilling resistant cucumber, known as 'NC76', and chilling susceptible cucumber, known as 'GY14'. Using 2% agarose gel electrophoresis, 145 SSR markers were identified as length variation markers between 'NC76' and 'GY14'. The SSR markers that showed no length polymorphism were then screened using high resolution melting analysis technique and additional 30 polymorphic SSR markers were identified. As a preliminary evaluation for mapping, 20 markers among these 175 markers were employed to a $F_2$ population of 'NC76' x 'GY14' cross. Linkage analysis revealed 13 markers that joined into six linkage groups and seven markers that remained unlinked. This result indicates that these 175 markers could be used for construction of a genetic map using a cross between 'NC76' and 'GY14' for further investigation in developing markers related to resistance to chilling in cucumbers.

• Journal of Plant Biotechnology
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• v.45 no.1
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• pp.9-16
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• 2018

The advent of available DNA barcoding technology has been extensively adopted to assist in the reference to differentiate the origin of various medicinal plants species. However, this technology is still far behind the curve of technological advances to be applied in a practical manner in the market to authenticate the counterfeit components or detect the contamination in the admixtures of medicinal plant species. Recently, a high resolution melting curve analysis technique was combined with the procedure of DNA barcoding (Bar-HRM) to accomplish this purpose. In this review, we tried to summarize the current development and bottleneck of processing related to the Bar-HRM technology for the practical application of medicinal plant species' differentiation in a viable global market. Although several successful results have been reported, there are still many obstacles to be resolved, such as limited number of DNA barcodes and single nucleotide polymorphisms, in particular, only one DNA barcode, internal transcribed sequence (ITS) of ribosomal DNA has been reported in the available nuclear genome. In addition, too few cases have been reported about the identification of counterfeit or contamination with processed medicinal plant products, in particular specifically the case of technology based infusion, jam and jelly products and components in which it is noted that DNA can be thereby degraded during the processing of these products and components.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.372-378
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• 2017

For comparison of the transcription profiles in apple (Malus domestica L.) cultivars differing in flesh color expression, two cDNA libraries were constructed. Differences in gene expression between red flesh apple cultivar, 'Redfield' and white flesh apple cultivar, 'Granny Smith' were investigated by next-generation sequencing (NGS). Expressed sequence tag (EST) of clones from the red flesh apple cultivar and white flesh apple cultivar were selected for nucleotide sequence determination and homology searches. High resolution melting (HRM) technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between red flesh apple cultivars and white flesh apple cultivars. We applied high resolution melting (HRM) analysis to discover single nucleotide polymorphisms (SNP) based on the predicted SNP information derived from the apple EST database. All 103 pairs of SNPs were discriminated, and the HRM profiles of amplicons were established. Putative SNPs were screened from the apple EST contigs by HRM analysis displayed specific difference between 10 red flesh apple cultivars and 11 white flesh apple cultivars. In this study, we report an efficient method to develop SNP markers from an EST database with HRM analysis in apple. These SNP markers could be useful for apple marker assisted breeding and provide a good reference for relevant research on molecular mechanisms of color variation in apple cultivars.

• Horticultural Science & Technology
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• v.28 no.4
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• pp.627-637
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• 2010

The consumption of tomato has greatly increased recently in Korea, and a large number of tomato cultivars are commercially available in the market. However, identification of tomato cultivars by morphological traits is extremely difficult because of the narrow genetic diversity of breeding lines. Therefore, it is necessary to develop molecular markers for cultivar identification in tomato. In this study, we surveyed single nucleotide polymorphism (SNP), and developed SNP marker sets for tomato cultivar identification. SNP markers were developed based on conserved ortholog set II (COSII) and intron-based markers derived from pepper EST sequences, and marker polymorphism was tested using high-resolution melting (HRM) analysis. A total of 628 primer sets was tested, and 417 primer sets amplifying single bands were selected. Of the 417 primer sets, 70 primer sets showing HRM polymorphism among 4 inbred lines were selected. Eleven markers were selected from the 70 primer sets and subjected to cultivar identification analysis. Thirty two commercial tomato cultivars were successfully identified using the marker set.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.5843-5846
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• 2015

Background: Detection of cervical high grade lesions in patients with atypical squamous cells of undetermined significance (ASCUS) is still a challenge. Our study tested the efficacy of the paired boxed gene 1 (PAX1) methylation analysis by methylation-sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in ASCUS and compared performance with the hybrid capture 2 (HC2) human papillomavirus (HPV) test. Materials and Methods: A total of 463 consecutive ASCUS women from primary screening were selected. Their cervical scrapings were collected and assessed by PAX1 methylation analysis (MS-HRM) and high-risk HPV-DNA test (HC2). All patients with ASCUS were admitted to colposcopy and cervical biopsies. The Chisquare test was used to test the differences of PAX1 methylation or HPV infection between groups. Results: The specificity, sensitivity, and accuracy for detecting CIN2 + lesions were: 95.6%, 82.4%, and 94.6%, respectively, for the PAX1 MS-HRM test; and 59.7%, 64.7%, and 60.0% for the HC2 HPV test. Conclusions: The PAX1 methylation analysis by MS-HRM demonstrated a better performance than the high-risk HPV-DNA test for the detection of high grade lesions (CIN2 +) in ASCUS cases. This approach could screen out the majority of low grade cases of ASCUS, and thus reduce the referral rate to colposcopy.

• Horticultural Science & Technology
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• v.31 no.1
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• pp.80-88
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• 2013

Carrot (Daucus carota L. var. sativa) is one of the most extensively used vegetable crops in the world and a significant source of nutrient because of its high content of ${\beta}$-carotene, well known as the precursor of vitamin A carotenoid. However, seed-hairs generated and elongated from the epidermal cell of seeds inhibit absorption and germination by various factors such as carotol and so on. Accordingly, mechanical hair removal process is essential before commercialization of carrot seeds. Because of this process, producers will have additional losses such as time consuming, manpower, capital and so on. Furthermore, physical damage of seeds causes irregular germination rate. To overcome such cumbersome weaknesses, new breeding program for developing hairless-seed carrot cultivar has been needed and studies for molecular markers related to seed-hair characteristic is needed for a new breeding program. Therefore, in this study, cDNA libraries from seeds of short-hair seed phenotype CT-SMR 616 OP 659-1 line, hairy-seed phenotype CT-SMR 616 OP 677-14 line and short-hair seed phenotype CT-ATR 615 OP 666-13 line, hairy-seed phenotype CT-ATR 615 OP 671-9 were constructed, respectively. Furthermore, 1,248 ESTs in each line, total 4,992 ESTs were sequenced. As a result, 19 SNP sites and 14 SNP sites in each of 2 combinations were confirmed by analyzing these EST sequences from short-hair and hairy-seed lines. Then we designed SNP primer sets from EST sequences of SNP sites for high resolution melting (HRM) analysis. Designed HRM primers were analyzed using hairy seed phenotype CT-SMR 616 OP 1040 line and short-hair seed phenotype CT-SMR 616 OP 1024, 1025, 1026 lines. One set of HRM primers showed specific difference between the melting curves of hairy and short-hair seed phenotype lines. Based on this result, allele-specific (AS) PCR primers were designed for easier selection between hairy-seed carrot and hairless seed carrot. These results of HRM and AS-PCR are expected to be useful in breeding of hairless seed carrot cultivar as a molecular marker.

• Journal of Plant Biotechnology
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• v.48 no.2
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• pp.71-76
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• 2021

Angelica is a perennial plant used widely for medicinal purposes. Information on the genetic diversity of Angelica populations is important for their conservation and germplasm utilization. Although Angelica is an important medicinal plant genus registered in South Korea, no molecular markers are currently available to distinguish individual species from other similar species in different countries, in particular, China and Japan. In this study, we developed single nucleotide polymorphism (SNP) markers derived from internal transcribed spacer regions of the nuclear ribosomal DNA to identify a distinct domestic species, Angelica gigas Nakai, via a high-resolution melting (HRM) curve analyses. We also performed HRM curve analysis of intentionally mixed genomic DNA samples from five Angelica species. Finally, we investigated A. gigas Nakai and A. sinensis using varying ratios of mixed genomic DNA templates. The SNP markers developed in this study are useful for rapidly identifying A. gigas species from different countries.

• Parasites, Hosts and Diseases
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• v.51 no.6
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• pp.645-650
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• 2013

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at $81.5{\pm}0.2^{\circ}C$, $79.0{\pm}0.3^{\circ}C$, $76.8{\pm}0.1^{\circ}C$, and $79.9{\pm}0.1^{\circ}C$, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.