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https://www.readbyqxmd.com/read/28101802/cold-pcr-technologies-in-the-area-of-personalized-medicine-methodology-and-applications
#1
REVIEW
Florence Mauger, Alexandre How-Kit, Jörg Tost
Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation...
January 18, 2017: Molecular Diagnosis & Therapy
https://www.readbyqxmd.com/read/28095454/non-homologous-end-joining-and-homology-directed-dna-repair-frequency-of-double-stranded-breaks-introduced-by-genome-editing-reagents
#2
Michail Zaboikin, Tatiana Zaboikina, Carl Freter, Narasimhachar Srinivasakumar
Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents...
2017: PloS One
https://www.readbyqxmd.com/read/28094303/genetic-polymorphisms-of-rs3077-and-rs9277535-in-hla-dp-associated-with-systemic-lupus-erythematosus-in-a-chinese-population
#3
Junlong Zhang, Wenli Zhan, Bin Yang, Anning Tian, Lin Chen, Yun Liao, Yongkang Wu, Bei Cai, Lanlan Wang
Although the SLE risk gene loci of HLA-DR and HLA-DQ within the major histocompatibility complex (MHC) region has been gradually revealed by recent Genome-Wide Association studies (GWAS), the association of HLA-DP polymorphisms with SLE was minimally reported. Considering that the variants in rs3077 and rs9277535 in the HLA-DP region could influence the immune response by affecting antigen presentation of HLA class II molecules to CD4(+) T cells, the present study aimed to explore the role of HLA-DP polymorphisms in SLE...
January 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28093071/spag6-and-l1td1-are-transcriptionally-regulated-by-dna-methylation-in-non-small-cell-lung-cancers
#4
Corinna Altenberger, Gerwin Heller, Barbara Ziegler, Erwin Tomasich, Maximilian Marhold, Thais Topakian, Leonhard Müllauer, Petra Heffeter, György Lang, Adelheid End-Pfützenreuter, Balazs Döme, Britt-Madeleine Arns, Walter Klepetko, Christoph C Zielinski, Sabine Zöchbauer-Müller
BACKGROUND: DNA methylation regulates together with other epigenetic mechanisms the transcriptional activity of genes and is involved in the pathogenesis of malignant diseases including lung cancer. In non-small cell lung cancer (NSCLC) various tumor suppressor genes are already known to be tumor-specifically methylated. However, from the vast majority of a large number of genes which were identified to be tumor-specifically methylated, tumor-specific methylation was unknown so far. Thus, the major aim of this study was to investigate in detail the mechanism(s) responsible for transcriptional regulation of the genes SPAG6 and L1TD1 in NSCLCs...
January 5, 2017: Molecular Cancer
https://www.readbyqxmd.com/read/28075457/high-resolution-melting-analysis-for-rapid-and-sensitive-notch1-screening-in-chronic-lymphocytic-leukemia
#5
Jing-Jing Xu, Fei-Rong Yao, Min Jiang, You-Tao Zhang, Feng Guo
Chronic lymphocytic leukemia (CLL) is a biological and clinical heterogeneous disease. Activating mutations of NOTCH1 have been implicated to be associated with adverse prognosis in CLL. The objective of the present study was to develop an effective high-resolution melting (HRM) assay for detecting NOTCH1 mutations. Genomic DNA (gDNA) extracted from 133 CLL patients was screened by HRM assay, and the results were compared with the data obtained using direct sequencing. The relative sensitivity of the HRM assay and direct sequencing was evaluated using diluted gDNA with different NOTCH1 mutational frequencies...
February 2017: International Journal of Molecular Medicine
https://www.readbyqxmd.com/read/28068926/screening-of-wt1-mutations-in-exon-8-and-9-in-children-with-steroid-resistant-nephrotic-syndrome-from-a-single-centre-and-establishment-of-a-rapid-screening-assay-using-high-resolution-melting-analysis-in-a-clinical-setting
#6
Annes Siji, Varsha Chhotusing Pardeshi, Shilpa Ravindran, Ambily Vasudevan, Anil Vasudevan
BACKGROUND: Mutations in Wilm's tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only mutation positive samples identified by HRM will be further processed for sequencing it will help in reducing the sequencing burden and speed up the screening process...
January 10, 2017: BMC Medical Genetics
https://www.readbyqxmd.com/read/28067764/high-resolution-temperature-measurement-of-liquid-stainless-steel-using-hyperspectral-imaging
#7
Wim Devesse, Dieter De Baere, Patrick Guillaume
A contactless temperature measurement system is presented based on a hyperspectral line camera that captures the spectra in the visible and near infrared (VNIR) region of a large set of closely spaced points. The measured spectra are used in a nonlinear least squares optimization routine to calculate a one-dimensional temperature profile with high spatial resolution. Measurements of a liquid melt pool of AISI 316L stainless steel show that the system is able to determine the absolute temperatures with an accuracy of 10%...
January 5, 2017: Sensors
https://www.readbyqxmd.com/read/28059095/h3f3-mutation-status-of-giant-cell-tumors-of-the-bone-chondroblastomas-and-their-mimics-a-combined-high-resolution-melting-and-pyrosequencing-approach
#8
Thibault Kervarrec, Christine Collin, Frédérique Larousserie, Corinne Bouvier, Sébastien Aubert, Anne Gomez-Brouchet, Béatrice Marie, Elodie Miquelestorena-Standley, Louis Romée Le Nail, Pierre Avril, Jean Christophe Pagès, Gonzague de Pinieux
Behjati et al recently described recurrent mutations of H3F3 genes in giant cell tumors of the bone and chondroblastomas. Both these entities belong to the spectrum of giant cell-rich bone lesions, often presenting a diagnostic challenge for the pathologist. Our aim was to investigate the value of searching for H3F3 mutations in the diagnosis of giant cell tumors of the bone and giant cell-rich chondroblastomas. Two hundred eighty-one bone lesion samples, including 170 giant cell tumors of the bone, 26 chondroblastomas and 85 other giant cell-rich and/or epiphyseal tumors, were analyzed...
January 6, 2017: Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc
https://www.readbyqxmd.com/read/28058700/xrcc1-arg399gln-gene-polymorphism-and-hepatocellular-carcinoma-risk-in-the-italian-population
#9
Concetta Santonocito, Margherita Scapaticci, Bojan Nedovic, Eleonora B Annicchiarico, Donatella Guarino, Emanuele Leoncini, Stefania Boccia, Antonio Gasbarrini, Ettore Capoluongo
BACKGROUND: The human X-ray repair cross-complementing protein 1 (XRCC1) gene encodes for one of the major repair factors involved in base excision repair (BER), which is reported to be associated with the risk of several cancers. A few studies have explored the association between risk of hepatocellular carcinoma (HCC) and single-nucleotide polymorphisms (SNPs) in different DNA repair genes, with contradictory results. The purpose of this study was to evaluate the association between XRCC1 Arg399Gln polymorphism and susceptibility to HCC...
January 2, 2017: International Journal of Biological Markers
https://www.readbyqxmd.com/read/28058013/aging-related-methylation-influences-the-gene-expression-of-key-control-genes-in-colorectal-cancer-and-adenoma
#10
Orsolya Galamb, Alexandra Kalmár, Barbara Kinga Barták, Árpád V Patai, Katalin Leiszter, Bálint Péterfia, Barnabás Wichmann, Gábor Valcz, Gábor Veres, Zsolt Tulassay, Béla Molnár
AIM: To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites. METHODS: In silico DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 (SFRP1) promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis...
December 21, 2016: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/28054984/detection-of-ribosomal-dna-sequence-polymorphisms-in-the-protist-plasmodiophora-brassicae-for-the-identification-of-geographical-isolates
#11
Rawnak Laila, Arif Hasan Khan Robin, Kiwoung Yang, Gyung Ja Choi, Jong-In Park, Ill-Sup Nou
Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study...
January 4, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28044290/microfluidic-dna-stretching-device-for-single-molecule-diagnostics
#12
Daisuke Onoshima, Yoshinobu Baba
The method described here enables the automatic stretching and patterning of single DNA molecules onto a solid surface. It does not require chemical modification of the DNA or surface modification of the substrate. To detect a signal variation caused by sequence-specific dye binding or partial melting, it is crucial that the DNA molecules are arrayed in a parallel direction inside the narrow microscopic field. The method uses zigzag-shaped microgrooves in a densely-arranged molecular patterning apparatus in a microfluidic channel...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28044264/clinicopathological-associations-of-k-ras-and-n-ras-mutations-in-indonesian-colorectal-cancer-cohort
#13
Michael Levi, Gintang Prayogi, Farid Sastranagara, Edi Sudianto, Grace Widjajahakim, Winiarti Gani, Albert Mahanadi, Jocelyn Agnes, Bela Haifa Khairunisa, Ahmad R Utomo
BACKGROUND: K-RAS and recently N-RAS gene mutation testing are mandatory requirements prior to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment of metastatic CRC. Mutation prevalence and distribution in Indonesian colorectal cancer (CRC) are not known. METHODS: Combined methods of PCR high-resolution melt (HRM), restriction fragment length polymorphism (RFLP), and direct DNA sequencing were used to genotype exons 2, 3, and 4 of both K-RAS and N-RAS genes for routine clinical testing of CRC patients...
January 3, 2017: Journal of Gastrointestinal Cancer
https://www.readbyqxmd.com/read/28042684/detection-of-the-myd88-mutation-by-the-combination-of-the-allele-specific-pcr-and-quenching-probe-methods
#14
S Nogami, N Kawaguchi-Ihara, E Shiratori, M Ohtaka, M Itoh, S Tohda
INTRODUCTION: The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR...
January 1, 2017: International Journal of Laboratory Hematology
https://www.readbyqxmd.com/read/28036310/comparison-of-hrm-analysis-and-three-rep-pcr-genomic-fingerprint-methods-for-rapid-typing-of-mrsa-at-a-brazilian-hospital
#15
Giovana Carolina Bodnar, Heloísa Moreira Martins, Caio Ferreira De Oliveira, Alexandre Tadachi Morey, Eliandro Reis Tavares, Juscélio Donizete Cardoso, Marcia Regina Eches Perugini, Lucy Megumi Yamauchi Lioni, Sueli Fumie Yamada-Ogatta, Renata Katsuko Takayama Kobayashi, Gerson Nakazato
INTRODUCTION: Infections caused by multidrug-resistant bacteria are increasingly common and represent a serious problem for public health. Staphylococcus aureus is one of the major agents of infections, and methicillin-resistant S. aureus (MRSA) has spread worldwide. The aim of this study was to phenotypically and genotypically characterize 55 MRSAs isolated in the University Hospital of Londrina, Paraná, Brazil, during 2010. METHODOLOGY: Bacterial isolates were characterized based on their antimicrobial susceptibility profile, biofilm production capacity, and staphylococcal chromosome cassette mec (SCCmec) type...
December 30, 2016: Journal of Infection in Developing Countries
https://www.readbyqxmd.com/read/28029290/high-resolution-melting-analysis-for-detection-of-a-single-nucleotide-polymorphism-and-the-genotype-of-the-myostatin-gene-in-warmblood-horses
#16
Priscila B S Serpa, Petra Garbade, Cláudio C Natalini, Ananda R Pires, Tainor M Tisotti
OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared...
January 2017: American Journal of Veterinary Research
https://www.readbyqxmd.com/read/28026816/-gstp1-apc-and-rassf1-gene-methylation-in-prostate-cancer-samples-comparative-analysis-of-ms-hrm-method-and-infinium-humanmethylation450-beadchip-beadchiparray-diagnostic-value
#17
L O Skorodumova, K A Babalyan, R Sultanov, A O Vasiliev, A V Govorov, D Y Pushkar, E A Prilepskaya, S A Danilenko, E V Generozov, A K Larin, E S Kostryukova, E I Sharova
There is a clear need in molecular markers for prostate cancer (PC) risk stratification. Alteration of DNA methylation is one of processes that occur during ÐÑ progression. Methylation-sensitive PCR with high resolution melting curve analysis (MS-HRM) can be used for gene methylation analysis in routine laboratory practice. This method requires very small amounts of DNA for analysis. Numerous results have been accumulated on DNA methylation in PC samples analyzed by the Infinium HumanMethylation450 BeadChip (HM450)...
November 2016: Biomedit︠s︡inskai︠a︡ Khimii︠a︡
https://www.readbyqxmd.com/read/28025791/high-resolution-melt-curve-analysis-based-on-methylation-status-for-human-semen-identification
#18
Caitlyn Fachet, Lawrence Quarino, K Joy Karnas
A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex(®) 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status...
December 27, 2016: Forensic Science, Medicine, and Pathology
https://www.readbyqxmd.com/read/28025302/micromechanics-of-sea-ice-frictional-slip-from-test-basin-scale-experiments
#19
Peter R Sammonds, Daniel C Hatton, Daniel L Feltham
We have conducted a series of high-resolution friction experiments on large floating saline ice floes in an environmental test basin. In these experiments, a central ice floe was pushed between two other floes, sliding along two interfacial faults. The frictional motion was predominantly stick-slip. Shear stresses, normal stresses, local strains and slip displacement were measured along the sliding faults, and acoustic emissions were monitored. High-resolution measurements during a single stick-slip cycle at several positions along the fault allowed us to identify two phases of frictional slip: a nucleation phase, where a nucleation zone begins to slip before the rest of the fault, and a propagation phase when the entire fault is slipping...
February 13, 2017: Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences
https://www.readbyqxmd.com/read/28024437/high-throughput-thermal-stability-assessment-of-dna-hairpins-based-on-high-resolution-melting
#20
Jing Wang, Ping Dong, Wei Wu, Xiaoming Pan, Xingguo Liang
On the basis of high-resolution melting, a high-throughput approach to measure melting temperatures (Tms) of short DNA hairpins was developed. With this method, Tms of thousands of triloop, tetraloop, and pentaloop hairpins involving various loop sequences and various closing base pairs (cbp) were obtained in hours. The stability of triloop hairpins decreased with the change of cbp (5'-3') in the order of c-g > g-c > t-a ≥ a-t, showing that the cbp of 5'-Pyr-Pur-3' (Pyr = pyrimidine, Pur = purine) contributed more stability than 5'-Pur-Pyr-3'...
December 26, 2016: Journal of Biomolecular Structure & Dynamics
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