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high resolution melt

Andrew Turner, Jurgen Sasse, Aniko Varadi
OBJECTIVES: Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements...
October 19, 2016: BMC Medical Genetics
Matthew S Payne, Demelza J Ireland, Rory Watts, Elizabeth A Nathan, Lucy L Furfaro, Matthew W Kemp, Jeffrey A Keelan, John P Newnham
BACKGROUND: Detection of Ureaplasma, Mycoplasma and Candida spp. in the vagina during pregnancy has previously been associated with preterm birth (PTB). However, the prevalence of these microorganisms and the associated obstetric risks (likely to be population-specific) have not been determined in Australian women; furthermore, in the case of Ureaplasma spp., very few studies have attempted characterisation at the species level and none have examined genotype/serovar status to further refine risk assessment...
October 18, 2016: BMC Pregnancy and Childbirth
Marta Ferro, Hada C Macher, Pilar Noguerol, Pilar Jimenez-Arriscado, Patrocinio Molinero, Juan M Guerrero, Amalia Rubio
Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias...
2016: Advances in Experimental Medicine and Biology
N García-Fernández, Hada C Macher, Amalia Rubio, Pilar Jiménez-Arriscado, C Bernal-Bellido, M L Bellido-Díaz, G Suárez-Artacho, Juan M Guerrero, M A Gómez-Bravo, Patrocinio Molinero
p53 is the most commonly mutated gene in malignant human cancers. To detect p53 mutations in circulating DNA (cirDNA) of transplanted hepatocellular carcinoma (HCC) patients could be an interesting approach to know of any tumor recurrence. In this study, our objective was to determine the utility of this method in the diagnosis and the prognosis of HCC tumor recurrence.Twenty four liver transplanted HCC patients were included in the study together with a group of healthy controls. Detection of the specific p53 mutation in cirDNA was performed by high-resolution melting PCR (HRM-PCR) and COLD-PCR immediately before the transplantation...
2016: Advances in Experimental Medicine and Biology
Ryan J Gallacher, Derek Keir, Nicholas Harmon, Graham Stuart, Sylvie Leroy, James O S Hammond, J-Michael Kendall, Atalay Ayele, Berhe Goitom, Ghebrebrhan Ogubazghi, Abdulhakim Ahmed
Melting of the mantle during continental breakup leads to magmatic intrusion and volcanism, yet our understanding of the location and dominant mechanisms of melt generation in rifting environments is impeded by a paucity of direct observations of mantle melting. It is unclear when during the rifting process the segmented nature of magma supply typical of seafloor spreading initiates. Here, we use Rayleigh-wave tomography to construct a high-resolution absolute three-dimensional shear-wave velocity model of the upper 250 km beneath the Afar triple junction, imaging the mantle response during progressive continental breakup...
October 18, 2016: Nature Communications
Siobhan Eustace Ryan, Fergus Ryan, Veronica O'Dwyer, Derek Neylan
PURPOSE: Approximately 95% of patients who are diagnosed with Leber's hereditary optic neuropathy (LHON) have one of three mitochondrial point mutations responsible for the disease, G3460A, G11778A, and T14484C. The purpose of this study was to develop a novel multiplex real-time amplification-refractory mutation system (ARMS) PCR combined with high-resolution melt curves to identify the individual mutations involved. The study aimed to provide a more robust, cost- and time-effective mutation detection strategy than that offered with currently available methods...
2016: Molecular Vision
Jezkova Eva, Kajo Karol, Zubor Pavol, Grendar Marian, Malicherova Bibiana, Mendelova Andrea, Dokus Karol, Lasabova Zora, Plank Lukas, Danko Jan
Breast cancer is a heterogeneous disease with very different responses to therapy and different length of survival. In many cases, however, the determination of the stage and histopathological characteristics of breast cancer is insufficient to predict prognosis and response to treatment for the vast heterogeneity of the disease. To understand the molecular signature of subtypes of breast cancer, we attempted to identify the methylation status of key tumour suppressor gene Ras association (RalGDS/AF-6) domain family member 1 isoform a (RASSF1A) and a member of the paired-like homeodomain transcription factor family which functions in left-right asymmetry development (PITX2) and to correlate results with known clinicopathological features of breast cancer...
October 15, 2016: Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine
T Kunicka, P Prochazka, I Krus, P Bendova, M Protivova, S Susova, V Hlavac, V Liska, P Novak, M Schneiderova, P Pitule, J Bruha, O Vycital, P Vodicka, P Soucek
BACKGROUND: This study addresses involvement of major 5-fluorouracil (5-FU) pathway genes in the prognosis of colorectal carcinoma patients. METHODS: Testing set and two validation sets comprising paired tumor and adjacent mucosa tissue samples from 151 patients were used for transcript profiling of 15 5-FU pathway genes by quantitative real-time PCR and DNA methylation profiling by high resolution melting analysis. Intratumoral molecular profiles were correlated with clinical data of patients...
October 12, 2016: BMC Cancer
Allison B Chambliss, Molly Resnick, Athena K Petrides, William A Clarke, Mark A Marzinke
BACKGROUND: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. METHODS: Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes...
October 12, 2016: Clinical Chemistry and Laboratory Medicine: CCLM
Stephen R Doyle, Samuel Armoo, Alfons Renz, Mark J Taylor, Mike Yaw Osei-Atweneboana, Warwick N Grant
BACKGROUND: Genetic surveillance of the human filarial parasite, Onchocerca volvulus, from onchocerciasis endemic regions will ideally focus on genotyping individual infective larval stages collected from their intermediate host, Simuliid blackflies. However, blackflies also transmit other Onchocerca species, including the cattle parasite O. ochengi, which are difficult to distinguish from the human parasite based on morphological characteristics alone. This study describes a versatile approach to discriminate between O...
October 10, 2016: Parasites & Vectors
Chloe Nguyen, Wei Ling Koh, Andrea Casteriano, Niek Beijerink, Christopher Godfrey, Graeme Brown, David Emery, Jan Šlapeta
BACKGROUND: Heartworm (Dirofilaria immitis) in dogs is considered endemic in Australia, but the clinical heartworm disease caused by the heartworm is rare and prevalence is low. The mainstream prevention of the heartworm is based on macrocyclic lactone (ML) administration. The aim of this study was to confirm endemism of the heartworm under current Australian conditions using a cohort of recent microfilaria-positive dogs which were on variable heartworm prevention. METHODS: A hotspot of canine heartworm antigen-positive and microfilaria-positive dogs has been detected recently in Queensland, Australia...
October 7, 2016: Parasites & Vectors
Yvonne Ukamaka Ajamma, Enock Mararo, David Omondi, Thomas Onchuru, Anne W T Muigai, Daniel Masiga, Jandouwe Villinger
Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes...
2016: F1000Research
Ming Song, Jingjian Li, Chao Xiong, Hexia Liu, Junsong Liang
Many members of the genus Artemisia are important for medicinal purposes with multiple pharmacological properties. Often, these herbal plants sold on the markets are in processed forms so it is difficult to authenticate. Routine testing and identification of these herbal materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use. In this study, five commonly used Artemisia species included Artemisia argyi, Artemisia annua, Artemisia lavandulaefolia, Artemisia indica, and Artemisia atrovirens were analyzed using high resolution melting (HRM) analysis based on the internal transcribed spacer 2 (ITS2) sequences...
October 4, 2016: Scientific Reports
Nariyoshi Matsumoto, Sayaka Mori, Hiroo Hasegawa, Daisuke Sasaki, Hayato Mori, Kazuto Tsuruda, Daisuke Imanishi, Yoshitaka Imaizumi, Tomoko Hata, Norihito Kaku, Kousuke Kosai, Naoki Uno, Yasushi Miyazaki, Katsunori Yanagihara
INTRODUCTION: Recently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60-80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. METHODS: We examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN...
November 1, 2016: Clinica Chimica Acta; International Journal of Clinical Chemistry
Miaoli Wu, Dan Rao, Yujun Zhu, Jing Wang, Wen Yuan, Yu Zhang, Ren Huang, Pengju Guo
BACKGROUND: Enterohepatic Helicobacter species (EHS) are widespread in rodent species around the world. Several studies have demonstrated that infection with EHS can interfere with the outcomes of animal experiments in cancer research and significantly influence the study results. Therefore, it is essential to establish a rapid detection and identification of EHS for biomedical research using laboratory rodents. Our study aimed to develop a rapid and sensitive method to detect and distinguish five enterohepatic Helicobacter species...
September 30, 2016: Helicobacter
Hevila Brognaro, Vitor Medeiros Almeida, Evandro Ares de Araujo, Vasily Piyadov, Maria Auxiliadora Morim Santos, Sandro Roberto Marana, Igor Polikarpov
The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure-function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans β-glycosidase from glycoside hydrolase family 1 (SdBgl1B). SdBgl1B has the optimum pH of 6.0 and a melting temperature T m of approximately 50 °C...
September 26, 2016: Molecular Biotechnology
Hirohide Takahashi, Atsushi Miyagi, Lorena Redondo-Morata, Simon Scheuring
With nanometer lateral and Angstrom vertical resolution, atomic force microscopy (AFM) has contributed unique data improving the understanding of lipid bilayers. Lipid bilayers are found in several different temperature-dependent states, termed phases; the main phases are solid and fluid phases. The transition temperature between solid and fluid phases is lipid composition specific. Under certain conditions some lipid bilayers adopt a so-called ripple phase, a structure where solid and fluid phase domains alternate with constant periodicity...
September 20, 2016: Small
Yang He, Li Zhong, Feifei Fan, Chongmin Wang, Ting Zhu, Scott X Mao
Amorphous materials are used for both structural and functional applications. An amorphous solid usually forms under driven conditions such as melt quenching, irradiation, shock loading or severe mechanical deformation. Such extreme conditions impose significant challenges on the direct observation of the amorphization process. Various experimental techniques have been used to detect how the amorphous phases form, including synchrotron X-ray diffraction, transmission electron microscopy (TEM) and Raman spectroscopy, but a dynamic, atomistic characterization has remained elusive...
October 2016: Nature Nanotechnology
Anjian Xu, Tingxia Lv, Bei Zhang, Wei Zhang, Xiaojuan Ou, Jian Huang
BACKGROUND: Wilson's disease (WD) is a rare autosomal recessive disorder characterized by the deposition of copper mainly in the liver or nerve system that leads to their dysfunction. Mutations in the gene encoding ATPase, Cu+ transporting, beta polypeptide (ATP7B) are causative for WD. The aim of this study was to develop a rapid and convenient assay for detection of the three most common causative ATP7B mutations, p.R778L, p.P992L, and p.V1106I. METHODS: Plasmids containing DNA fragments harboring each of the three ATP7B mutations were constructed...
September 17, 2016: Journal of Clinical Laboratory Analysis
Marianeli Rodriguez, Nilufer Yesilirmak, Priyanka Chhadva, Brian Goldhagen, Carol Karp, Anat Galor
PURPOSE: To evaluate whether high-resolution optical coherence tomography (HR-OCT) can aid in differentiation of inflammatory versus noninflammatory causes of peripheral corneal thinning. METHODS: Retrospective chart review of 10 patients with peripheral corneal thinning and their respective slit-lamp photographs and HR-OCT images. RESULTS: Ten patients were identified who had peripheral corneal thinning and HR-OCT images. Five had a clinical history consistent with Terrien marginal degeneration (TMD), whereas 5 had thinning believed to be inflammatory in origin...
September 14, 2016: Cornea
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