• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6619-6623
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• 2013

Background:The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. Methods: High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. Results: The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Conclusions: Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

• BMB Reports
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• 제55권11호
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• pp.553-558
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• 2022

Hepatocellular carcinoma (HCC) is dangerous cancer that often evades early detection because it is asymptomatic and an effective detection method is lacking. For people with chronic liver inflammation who are at high risk of developing HCC, a sensitive detection method for HCC is needed. In a meta-analysis of The Cancer Genome Atlas pan-cancer methylation database, we identified a CpG island in the USP44 promoter that is methylated specifically in HCC. We developed methylation-sensitive high-resolution melting (MS-HRM) analysis to measure the methylation levels of the USP promoter in cell-free DNA isolated from patients. Our MS-HRM assay correctly identified 40% of patients with early-stage HCC, whereas the α-fetoprotein test, which is currently used to detect HCC, correctly identified only 25% of early-stage HCC patients. These results demonstrate that USP44 MS-HRM analysis is suitable for HCC surveillance.

• 한국수산과학회지
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• 제47권3호
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• pp.241-246
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• 2014

Viruses in the genus Megalocytivirus have been subdivided into four subgroups. Among these subgroups 2 and 4, represented by the red sea bream iridovirus (RBIV) and the olive flounder iridovirus (FLIV), respectively, are non-exotic. subgroups 1 and 3, represented by the red sea bream iridovirus (RSIV) and the infectious spleen and kidney necrosis virus (ISKNV), respectively, have not been detected in Korea and are known as exotic. Shellfish are filter-feeders, and can thus filter and accumulate Megalocytivirus in their digestive glands, allowing us to track viral contamination in surrounding aquatic environment. In this study, we developed a high-resolution melting (HRM) analysis to differentiate among subgroups of Megalocytivirus accumulated in shellfish, and confirmed the convenience and efficiency of this method. More than two subgroups of Megalocytivirus were found in the digestive gland of a single shellfish. We classified all Megalocytivirus viruses from shellfish in Korea into subgroups 2 and 4, although proportions of subgroups were different among regions. Compared to nucleotide sequencing analysis, HRM analysis is a simple and rapid method for differentiating of Megalocytivirus subgroups.

• Parasites, Hosts and Diseases
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• 제51권6호
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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.

• 한국약용작물학회지
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• 제23권6호
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• pp.439-445
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• 2015

Background : Correct identification of Panax species is important to ensure food quality, safety, authenticity and health for consumers. This paper describes a high resolution melting (HRM) analysis based method using internal transcribed spacer (ITS) and 5.8S ribosomal DNA barcoding regions as target (Bar-HRM) to obtain barcoding information for the major Panax species and to identify the origin of ginseng plant. Methods and Results : A PCR-based approach, Bar-HRM was developed to discriminate among Panax species. In this study, the ITS1, ITS2, and 5.8S rDNA genes were targeted for testing, since these have been identified as suitable genes for use in the identification of Panax species. The HRM analysis generated cluster patterns that were specific and sensitive enough to detect small sequence differences among the tested Panax species. Conclusion : The results of this study show that the HRM curve analysis of the ITS regions and 5.8S rDNA sequences is a simple, quick, and reproducible method. It can simultaneously identify three Panax species and screen for variants. Thus, ITS1HRM and 5.8SHRM primer sets can be used to distinguish among Panax species.

• 생명과학회지
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• 제21권3호
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• pp.468-472
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• 2011

DNA 마커는 유전현상 분석이나 품종육성에 널리 사용되고 있다. 본 연구에서는 내냉성 오이 계통인 'NC76'과 냉해 감수성 계통인 'GY14'로부터 내냉성 연관 마커을 목적으로 기존의 총 995개 SSR 마커의 다형성을 평가하였다. Agarose gel 전기영동법으로 'NC76과 'GY14' 간 PCR증폭 산물의 길이 다형성을 보이는 145개 SSR 마커를 개발하였으며, high resolution melting (HRM) 기술을 사용하여 염기서열 다형성을 보이는 30개의 SSR 마커를 확인하였다. 개발된 175개 SSR 마커 중 20개 마커를 선발하여 'NC76'과 'GY14' 간 $F_2$ 분리 집단에 대한 연관지도를 작성하였으며 그 결과 13개의 마커가 예상했던 연관군에 일치하여 위치됨을 확인할 수 있었다. 따라서 본 연구에서 확인된 175개의 SSR 마커는 향 후 냉해 저항성 연관 마커 개발을 위한 오이 유전자 지도 작성 및 이를 통한 품종 육성에 크게 활용될 수 있을 것으로 기대된다.

• Parasites, Hosts and Diseases
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• 제51권6호
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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.

• 대한원격탐사학회지
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• 제30권6호
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• pp.809-816
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• 2014

Ocean Color product들은 해양 생태계를 이해하기 위해 중요한 변수이다. 고위도 지역에서는 해빙이 바다로 유입되어 ocean color product에 광학적인 영향을 미친다. 본 연구에서는 북극 다산기지 근해의 피요르드에 대한 광학적 특성을 평가하고 높은 공간해상도를 가진 Landsat-8 OLI 영상의 엽록소-a(chlorophyll-a)와 부유물질(suspended sediment) 농도를 산출하고자 한다. 엽록소-a와 부유물질 농도를 추정하기 위해서 band ratio를 이용한 다양한 회귀 모델을 테스트했다. 위성영상과 관측된 실측 값과의 시간적인 차이 때문에 사용된 회귀모델은 높은 상관관계를 가지지는 못하였다. 하지만 Landsat-8 OLI 영상을 이용한 모델에서 생산된 엽록소-a와 부유물질 농도는 북극해 주변 피요르드와 해안지역에 대한 고해상도 위성 데이터를 활용한 모니터링 가능성을 보여주었다. 북극해 주변의 기후변화 패턴을 이해하기 위해서는 해양 생태계 변화에 ice meltig이 어떠한 영향을 미치는지를 이해하는 것이 필요하다. 본 연구의 결과는 고위도지역에서 ice melting이 해양생태계 변화에 미치는 영향을 고해상도로 모니터링을 하는데 사용된다. 극지연구소는 2002년부터 스발바드 다산기지을 운영하고 있으며 한국의 북극 기지를 기반으로 연구를 수행하였다.

• 환경생물
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• 제40권2호
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• pp.199-205
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• 2022

Two tree frogs, Dryophytes suweonensis and Dryophytes japonicus, inhabiting Korea, are morphologically similar and share the same habitats. Therefore, they are identified mainly through their calls, especially for males. Dryophytes suweonensis is registered as an endangered (IUCN: EN grade) and protected species in South Korea. Thus, it is necessary to develop a method to rapidly identify and discriminate the two species and establish efficient protection and restoration plans. We identified significant genetic variation between them by sequencing a maternally-inherited mitochondrial 12S ribosomal DNA region. Based on the sequence data, we designed a pair of primers containing 7bp differences for high resolution melting(HRM) analysis to rapidly and accurately characterize their genotypes. The HRM analysis using genomic DNA showed that the melting peak for D. suweonensis was 76.4±0.06℃, whereas that of D. japonicus was 75.0±0.05℃. The differential melt curve plot further showed a distinct difference between them. We also carried out a pilot test for the application of HRM analysis based on immersing D. suweonensis in distilled water for 30 min to generate artificial environmental DNA(eDNA). The results showed 1.10-1.31℃ differences in the melting peaks between the two tree frog samples. Therefore, this HRM analysis is rapid and accurate in identifying two tree frogs not only using their genomic DNA but also using highly non-invasive eDNA.

• 한국균학회지
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• 제50권3호
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• pp.225-233
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• 2022

Pyogo (Shiitake, Lentinula edodes) is one of the most important edible mushrooms because of its outstanding nutritive and medicinal value. In the registration and protection procedure for newly developed mushroom cultivars, the application of molecular markers that can supplement the morphological characteristic-based distinction has been strongly requested. Sanjo 701 and Chamaram, newly developed at the Federation Forest Mushroom Research Center of Korea, have been characterized as innovative cultivars suitable for customer demands because of their high yields and cultivation rates. However, no technical tools can protect the rights to these important cultivars. In this study, using comparative genomic information from 23 commercially available pyogo cultivars, we identified single nucleotide polymorphisms (SNPs) that accurately differentiated Sanjo701 and Chamaram from the other cultivars. We also developed high-resolution melting analysis (HRM)-based SNP markers that discriminate among the tested 23 pyogo cultivars. The developed SNP markers can be utilized for rapid, accurate identification of pyogo cultivars with low genetic diversity and to prevent cultivar contamination caused by illegally distributed inocula. In addition, these markers can serve as a crucial scientific basis for securing the right to conserve new cultivars in international markets.