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High resolution melting

Ollin Celeste Martínez-Ramírez, Rebeca Pérez-Morales, Clementina Castro-Hernández, Maria Eugenia Gonsebatt, Leonora Casas-Ávila, Margarita Valdés-Flores, Pavel Petrosyan, Valeria Ponce de León-Suárez, Julieta Rubio
O6 -Methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs the DNA damage caused by the tobacco habit, and low activity of this enzyme has been associated with a risk of lung cancer (LC). Our objective was to determine the association of the promoter methylation and the rs12917 polymorphism of MGMT with formation of DNA bulky adducts and the risk of LC in the Mexican Mestizo population. In this study are included 431 subjects. High-resolution melting analysis was used to determine the polymorphism MGMT rs12917 and methylation levels...
February 20, 2019: DNA and Cell Biology
Sukumar Biswas, Rong Li, Zheng Yuan, Dabing Zhang, Xiangxiang Zhao, Jianxin Shi
Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations...
February 19, 2019: Plant Cell Reports
Carlos Valério Rocha-Junior, Michele Gomes Da Broi, Cristiana Libardi Miranda-Furtado, Paula Andrea Navarro, Rui Alberto Ferriani, Juliana Meola
Endometriosis is frequently related to infertility and little is known about the mechanisms underlying this association. Some studies point to an endometrial factor involved in this condition, which could compromise embryo implantation. Progesterone plays crucial role in endometrial receptivity by acting through progesterone receptor ( PGR) isoforms PR-A and PR-B whose expression is epigenetically regulated by DNA methylation, in a specific promoter region for each isoform. Epigenetic changes in PGR-A and PGR-B may be related to progesterone resistance of endometriosis-related infertility...
February 19, 2019: Reproductive Sciences
L A McMurdie, A K Rowe, R A Houze, S R Brodzik, J P Zagrodnik, T M Schuldt
Enhancement of precipitation processes aloft over complex terrain is documented using reflectivity data from an S-band scanning radar (NPOL) that was deployed on the west coast of Washington State during the Olympic Mountains Experiment (OLYMPEX). From November 2015 through mid-January 2016, NPOL obtained high-resolution data within sectors over the ocean and over the windward slopes of the Olympic Mountains. Contoured Frequency by Altitude Diagrams of radar reflectivity highlight a higher frequency of occurrence of larger reflectivities for all heights between 2 and 8 km over land compared to ocean, with the largest difference in the 4- to 6-km range indicating a robust signature of enhancement aloft over the windward slopes...
November 16, 2018: Journal of Geophysical Research. Atmospheres: JGR
Lucia Landi, Sergio Murolo, Gianfranco Romanazzi
'Candidatus Phytoplasma solani' is the causal agent of Bois noir (BN) in grapevine (Vitis vinifera). It is usually detected in leaves, where typical disease symptoms are seen. However, little information is available on the presence of this phytoplasma in grapevine roots. Here, we investigated 'Ca. P. solani' in roots collected from 28 symptomatic, 27 recovered and eight asymptomatic grapevine plants. Protocols based on high-resolution melting (HRM) combined with real-time quantitative PCR (qPCR-HRM) and nested-qPCR-HRM were developed to identify 'Ca...
February 14, 2019: Scientific Reports
Helena Čelešnik, Uroš Potočnik
We report two restriction enzyme-based approaches for generating clean locus-specific unmethylated controls for methylation-sensitive high-resolution melting (MS-HRM) analyses. These unmethylated standards are derived from DNA treated with the demethylating agent 5-aza-2-deoxycytidine (5-Aza-dc). By using them, we overcome a limitation of 5-Aza-dc treatment - incomplete demethylation at various genomic regions. When 5-Aza-dc-treated DNA is used directly as unmethylated MS-HRM standard, partially demethylated DNA can give false methylation results...
February 14, 2019: BioTechniques
Youwei Jiang, Bingqing Luo, Xing Cheng
Thermoplastic polymer micro- and nanostructures suffer pattern decay when heated to a temperature close to or above the polymer's glass transition temperature. In this work, we report enhanced thermal stability of polycarbonate nanostructures at temperatures well above their glass transition temperatures. Based on this observation, we develop a unique technique for high-resolution polymer patterning by polymer reflows. This technique is characterized as the precise control of polymer reflows regardless of the annealing time, which avoids the time-domain nonlinear reflow of the polymer melt...
February 12, 2019: Materials
Taimour Langaee, Nihal El Rouby, Lynda Stauffer, Cheryl Galloway, Larisa H Cavallari
AIMS: This study was designed to develop a high-resolution melting (HRM) analysis-based cardiovascular (CV) pharmacogenetics (PGx) genotyping panel for the Canon DNA Genetic Analyzer multiplex genotyping platform and cross-validate its performance with the TaqMan® -based OpenArray® method. METHODS: The CV PGx genotyping panel containing 17 single nucleotide polymorphisms (SNPs) selected from 5 genes (CYP2C9, CYP2C19, CYP4F2, SLCO1B1, and VKORC1) and the CYP2C cluster was used to compare genotyping results between analysis methods...
February 13, 2019: Genetic Testing and Molecular Biomarkers
Tangyuheng Liu, Jiajia Song, Mei Zhang, Siyue Li, Jinya Zhang, Xuejiao Hu, Zhenzhen Zhao, Wu Peng, Qian Wu, Hao Bai, Yinyu Li, Xiaojun Lu, Binwu Ying
BACKGROUND: We investigated the relationship between hepatitis B virus (HBV)-related pathogenesis and single nucleotide polymorphisms (SNPs) in interleukin-21 (IL-21)-JAK-STAT signaling pathway genes. METHODS: We used the high-resolution melting (HRM) method to genotype five SNPs (IL-21 rs2221903, IL-21 rs4833837, IL-21 receptor (IL-21R) rs2285452, JAK3 rs3008, and STAT3 rs1053023) in 546 HBV-infected patients and 353 healthy Chinese subjects. The HBV-infected patients were further divided into subgroups based on the HBV-related pathologies: chronic hepatitis B (CHB), HBV-related liver cirrhosis (LC), and HBV-related hepatocellular carcinoma (HCC)...
February 13, 2019: Journal of Clinical Laboratory Analysis
Patricia Landolt, Roger Stephan, Simone Scherrer
The rapid identification and differentiation of members of the Mycobacterium tuberculosis complex (MTBC) is essential to assess the potential zoonotic risk. Different available molecular methods are time consuming since they depend on cultivation of mycobacteria. High Resolution Melting (HRM) is a low cost, rapid and easy to perform single-tube method not limited to cultured samples. In this study, a HRM assay specifically targeting gyrB was developed to simultaneously identify and differentiate Mycobacterium (M...
February 12, 2019: Scientific Reports
Roshanak Aboutorabi, Ehsan Behzadi, Mohammad Javad Sadegh, Seyed Pooriya Fatehi, Soroosh Semsarzadeh, Yasaman Zarrin, Mohammad Kazemi, Laleh Rafiee, Fatemeh Sadat Mostafavi
Background: According to the literature review, polymorphisms of tumor necrosis factor alpha's (TNF-α) promoter region are probably the genetic risk factors of recurrent pregnancy loss. This study has investigated five single nucleotide polymorphisms in the TNF-α gene's promoter region to evaluate their relationship with recurrent pregnancy loss disorder. Methods: Blood samples were taken from 65 women with recurrent pregnancy loss (Case group) and 65 healthy women with a history of successful pregnancy (Control group)...
October 2018: Journal of Reproduction & Infertility
Julio Novoa, Karem Chokmani, Rachid Lhissou
Riparian strips are used worldwide to protect riverbanks and water quality in agricultural zones because of their numerous environmental benefits. A metric called Riparian Strip Quality Index, which is based on the percentage area of riparian vegetation, is used to evaluate their ecological condition. This index measures the potential capacity of riparian strips to filter sediments, retain pollutants, and provide shelter for terrestrial and aquatic species. This research aims to improve this metric by integrating the ability of riparian strips to intercept surface runoff, which is the major cause of water pollution and erosion in productive areas...
December 10, 2018: Science of the Total Environment
Hersh A Ham-Karim, Henry Okuchukwu Ebili, Kirsty Manger, Wakkas Fadhil, Narmeen S Ahmad, Susan D Richman, Mohammad Ilyas
BACKGROUND: Mutation testing in the context of neoadjuvant therapy must be performed on biopsy samples. Given the issue of tumour heterogeneity, this raises the question of whether the biopsies are representative of the whole tumour. Here we have compared the mutation profiles of colorectal biopsies with their matched resection specimens. METHODS: We performed next-generation sequencing (NGS) analysis on 25 paired formalin-fixed, paraffin-embedded colorectal cancer biopsy and primary resection samples...
February 11, 2019: Molecular Diagnosis & Therapy
Enderson Murillo, Carlos Muskus, Luz A Agudelo, Iván D Vélez, Freddy Ruiz-Lopez
Among vector-borne diseases malaria is the leading cause of morbidity in the world, with more than 200 million cases per year and a large number of deaths. The techniques traditionally used for the detection of Plasmodium in humans and Anopheles mosquitoes include microscopy, IRMA, ELISA, antibody or molecular assays, and anopheline dissection. However, these techniques are limited by their requirement of skilled personnel, low sensitivity or long processing times. A PCR-based high-resolution melting (PCR-HRM) analysis was developed for the detection and identification of P...
February 8, 2019: Scientific Reports
Judith Rittenschober-Böhm, Thomas Waldhoer, Stefan M Schulz, Birgit Pimpel, Katharina Goeral, David C Kasper, Armin Witt, Angelika Berger
BACKGROUND: Ureaplasma species (spp.) are the bacteria most often isolated from the amniotic cavity of women with preterm labor or preterm premature rupture of membranes, thus, the link between intrauterine Ureaplasma spp. infection and adverse pregnancy outcome is clearly established. However, as vaginal Ureaplasma spp. colonization is very common in pregnant women, it is not clear why these microorganisms cause ascending infections in some cases but remain asymptomatic in most pregnancies...
February 5, 2019: American Journal of Obstetrics and Gynecology
Jing Dong, Qinfeng Xu, Chen-Chen Li, Chun-Yang Zhang
We develop a single-color multiplexing strategy by the integration of high-resolution melting pattern recognition with loop-mediated isothermal amplification (LAMP). This strategy can identify multiple amplicons with a small DNA melting temperature (Tm) difference (∼0.2 °C) without the involvement of either multicolor labels or parallelized multiplexing, and it can sensitively detect LAMP amplicons with the initial DNA concentrations ranging from 10 to 108 copies.
February 8, 2019: Chemical Communications: Chem Comm
Saumu Pazia Juma, Athanasia Maro, Suporn Pholwat, Stellah G Mpagama, Jean Gratz, Alphonse Liyoyo, Eric R Houpt, Gibson S Kibiki, Blandina T Mmbaga, Scott K Heysell
BACKGROUND: Tuberculosis (TB) is the leading cause of death from an infectious disease and the roll-out of rapid molecular diagnostics for rifampin resistance has resulted in a steady rise in the number of patients with multidrug-resistant (MDR)-TB referred for treatment. Pyrazinamide is used in susceptible TB treatment for 6 months when used in combination with rifampin, isoniazid and ethambutol and is an important companion drug in novel MDR-TB trials. This study was undertaken to determine the prevalence of pyrazinamide resistance by either phenotypic or pncA testing among patients admitted to a referral hospital in Tanzania for drug-susceptible and MDR-TB treatment...
February 7, 2019: BMC Infectious Diseases
Avinash Kumar, Simmi P Sreedharan, Parvatam Giridhar
Gene families, like the conserved transcription factor families, evolve through gene duplications and share moderate similarity between member genes. Lack of genomic data makes it difficult to design high-specificity primers to the target genes. Furthermore, many primers under-perform in highly sensitive assays like quantitative PCR due to issues of thermodynamic nature, thereby increasing the cost and time for analysis. A methodology involving intra-species and inter-generic bioinformatic sequence comparison combined with thermodynamic estimation of primer performance was used for one-time design of gene specific primers for different WRKYs, Mitogen Activated Protein-kinases and N-methyltransferases of Coffea canephora without the aid of genome sequence resources...
February 6, 2019: Molecular Biology Reports
Sónia Soares, Liliana Grazina, Isabel Mafra, Joana Costa, M Alice Pinto, M Beatriz P P Oliveira, Joana S Amaral
Honey is the natural sweet substance produced by Apis mellifera honeybees in Europe. Depending on the country/region, the A. mellifera subspecies native to Europe belong to three different lineages: A (A. m. iberiensis), M (A. m. iberiensis and A. m. mellifera) and C (A. m. ligustica and A. m. carnica). In this work, two DNA-based approaches were developed with the aim of entomological authentication of European honeys. A cytb specific PCR assay was proposed to identify A-lineage honeybees, while a second method based on real-time PCR coupled to high resolution melting analysis targeting the COI gene was developed to differentiate C- and M-lineages honeybees...
June 15, 2019: Food Chemistry
Anastasia Koltsakidou, Zoi Terzopoulou, George Z Kyzas, Dimitrios N Bikiaris, Dimitra A Lambropoulou
In the present study, polymer supported nanocomposites, consisting of bio-based poly(ethylene furanoate) polyester and TiO₂ nanoparticles, were prepared and evaluated as effective photocatalysts for anti-inflammatory/analgesic drug removal. Nanocomposites were prepared by the solvent evaporation method containing 5, 10, 15, and 20 wt% TiO₂ and characterized using Fourier Transform Infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM)...
February 4, 2019: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
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