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Melting Curve Analysis (melting + curve_analysis)
Selected AbstractsReal Time Electrochemical Monitoring of DNA/PNA Dissociation by Melting Curve AnalysisELECTROANALYSIS, Issue 14 2009Xiaoteng Luo Abstract An immobilization-free electrochemical method is reported for real-time monitoring of the DNA hybrid dissociation between a ferrocene labeled peptide nucleic acid (PNA) and a fully-complementary or single-base-mismatched DNA. This method takes advantages of electrostatic charge characteristics and interactions among the neutrally charged PNA, the negatively charged DNA and the negatively charged electrode surface made of indium tin oxide (ITO). When a ferrocene labeled PNA (Fc-PNA) sequence is hybridized to a complementary DNA strand, electrostatic repulsion between the negatively charged PNA/DNA hybrid and the negative ITO surface retards the diffusion of the electroactive Fc to the electrode, resulting in a much reduced electrochemical signal. On the other hand, when the Fc-PNA is dissociated from the hybrid at elevated temperatures, the neutrally charged Fc-PNA easily diffuses to the electrode with an enhanced electrochemical signal. Therefore, an electrochemical melting curve of the Fc-PNA/DNA hybrid can be obtained by measuring the Fc signal with the increasing temperature. This strategy allows monitoring of the dissociation of the DNA hybrid in real time, which might lead to a simple detection method for single nucleotide polymorphism (SNP) analysis. [source] Rapid diversification of measles virus genotypes circulating in Morocco during 2004,2005 epidemics,JOURNAL OF MEDICAL VIROLOGY, Issue 11 2006Amal Alla Abstract Measles virus strains circulating in six different regions in Morocco during 2004,2005 were analysed. They were genotyped using two different methods: the recently developed method based on real-time PCR amplification and melting curve analyses, and the conventional method based on nucleic acid sequencing and phylogenetic analysis of 456 nucleotides of the 3,-region of the nucleoprotein (N) gene sequence. Five genotypes (A, B3.2, C2, D7 and D8) were shown to be circulating during this period. Previous studies on measles virus genotypes in Morocco (1998,2003) showed that only the genotype C2 was present and was considered to be endemic. Sequence comparison of the 2004,2005 viruses with other measles strains suggests that measles strains belonging to genotype B3.2 were probably imported from West Africa, whereas those belonging to genotypes D7 and D8 were imported from Europe. These studies which identify the route of importation of measles are important for developing strategies for measles elimination in Morocco. J. Med. Virol. 78:1465,1472, 2006. © 2006 Wiley-Liss, Inc. [source] Association of the TRAF1/C5 locus with increased mortality, particularly from malignancy or sepsis, in patients with rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 1 2009Vasileios F. Panoulas Objective Recent genome-wide association studies have identified TRAF1/C5 as a rheumatoid arthritis (RA) susceptibility locus. Tumor necrosis factor receptor,associated factor 1 (TRAF1) has been implicated in the regulation of antiapoptotic pathways, whereas C5 has a well-established role in defense against infection. The purpose of this study was to examine the association of the TRAF1/C5 locus with death in patients with RA. Methods Genomic DNA samples were collected from a prospective cohort of 400 RA patients. TRAF1/C5 rs3761847 was identified using real-time polymerase chain reaction and melting curve analyses. The association of TRAF1/C5 rs3761847 alleles with the risk of death was assessed using Cox proportional hazards regression analyses. Results TRAF1/C5 rs3761847 GG homozygote status was associated with an increased risk of death (hazard ratio 3.96 [95% confidence interval 1.24,12.6], P = 0.020) as compared with AA homozygote status. The excess mortality was attributed to deaths due to malignancies and sepsis but not cardiovascular disease (CVD). This polymorphism was one of the strongest predictors of death in RA (for TRAF1/C5 GG versus AA, hazard ratio 3.85 [95% confidence interval 1.18,12.59], P = 0.026) alongside the erythrocyte sedimentation rate, triglyceride level, prednisolone use, and age. Conclusion The risk of death in RA is increased in TRAF1/C5 rs3761847 GG homozygotes and appears to be independent of RA activity and severity as well as comorbidities relevant to CVD. If this finding is replicated in future studies, TRAF1/C5 genotyping could identify patients at increased risk of death, particularly death due to malignancy or sepsis. [source] Cover Picture: Electrophoresis 14'09ELECTROPHORESIS, Issue 14 2009Article first published online: 28 JUL 200 Issue no. 14 is an Emphasis Issue with 9 articles on various aspects of "Proteins and Proteomics" while the remaining 14 articles are arranged into 4 different parts including "Microfluidics and Miniaturization", "Genotyping and Transcriptomics", "Enantioseparations", and "Nanoparticles and Abused Drugs Analyses". Selected articles are: Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresis ((10.1002/elps.200900026)) 2-D difference in gel electrophoresis combined with Pro-Q Diamond staining: A successful approach for the identification of kinase/phosphatase targets ((10.1002/elps.200800780)) Microvalves actuated sandwich immunoassay on an integrated microfluidic system ((10.1002/elps.200800818)) Chemical gradient-mediated melting curve analysis for genotyping of SNPs ((10.1002/elps.200800729)) [source] Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis,HUMAN MUTATION, Issue 6 2009Tú Nguyen-Dumont Abstract Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been reported. Here, we show that it can improve the sensitivity and the efficiency of large-scale case,control mutation screening. Two exons of ATM, both containing an SNP interfering with standard mutation scanning, were selected for screening of 1,356 subjects from an international breast cancer genetics study. Asymmetric PCR was performed in the presence of an SNP-specific unlabeled probe. Stratification of the samples according to their probe-target melting was aided by customized HR-melt software. This approach improved identification of rare known and unknown variants, while dramatically reducing the sequencing effort. It even allowed genotyping of tandem SNPs using a single probe. Hence, HR-melt is a rapid, efficient, and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.Hum Mutat 30:1,7, 2009. © 2009 Wiley-Liss, Inc. [source] Myeloperoxidase (MPO) genotype and lung cancer histologic types: The MPO ,463 A allele is associated with reduced risk for small cell lung cancer in smokersINTERNATIONAL JOURNAL OF CANCER, Issue 5 2002Heike Dally Abstract MPO participates in the metabolic activation of tobacco carcinogens such as PAHs. A frequent MPO ,463 G,A polymorphism in the promoter region reduces MPO transcription and has been correlated with >4-fold lower benzo[a]pyrene,DNA adduct levels in the skin of coal tar,treated patients. Four of 7 case-control studies found significantly reduced lung cancer risk associated with the A allele. Due to their different etiologies, we examined whether the MPO genotype affects histologic lung cancer types differentially. A case-control study was conducted in 625 ever-smoking lung cancer patients, including 228 adenocarcinomas, 224 SCCs, 135 SCLCs and 340 ever-smoking hospital controls. MPO genotyping was performed by capillary PCR followed by fluorescence-based melting curve analysis. Combining the MPO ,463 (G/A+A/A) genotypes, a protective effect approaching significance (OR = 0.75, 95% CI 0.55,1.01) was observed when comparing all lung cancer cases to controls. Among histologic types of lung cancer, a weak protective effect was found for both adenocarcinoma (OR = 0.81, CI 0.55,1.19) and SCC (OR = 0.82, CI 0.56,1.21); a stronger and significant effect was found for SCLC (OR = 0.58, CI 0.36,0.95; p = 0.029). Our results also suggest that the MPO genotype varies among inflammatory nonmalignant lung diseases. In conclusion, our results emphasize the need for a separate analysis of lung cancer histologic types and an adjustment for inflammatory nonmalignant lung diseases in future MPO-related studies. We confirm that the MPO ,463 A variant affords a protective effect against lung cancer risk in smokers, which was strongest for SCLC patients. © 2002 Wiley-Liss, Inc. [source] Prevalence of risk factors for statin-induced myopathy in rheumatoid arthritis patientsMUSCULOSKELETAL CARE, Issue 1 2010Tracey E. Toms MBChB MRCP Abstract Objectives:,Statins are widely prescribed in patients with rheumatoid arthritis (RA). Although statins offer overwhelming cardiovascular benefits, their use can be associated with the development of a statin-induced myopathy. Several factors increase the risk of developing statin-induced myopathy, including the single nucleotide polymorphism (SNP) rs4149056, located within the gene encoding solute carrier organic anion transporter (SLCO1B1). We aimed to identify the frequency of risk factors for statin-induced myopathy and establish whether the rs4149056 genotype is more prevalent in RA. Methods:,A total of 396 RA patients and 438 non-RA controls were studied. DNA samples were obtained from all patients. The SNP rs4149056 was identified using real-time polymerase chain reaction and melting curve analysis. Genotypic and allelic frequencies were calculated using the chi-squared test. Results:,Almost 80% of RA patients had one or more risk factor (range 1,5) for the development of statin-induced myopathy. Of the 74 RA patients treated with statins, 90% had one or more (range 1,4) risk factors. No differences in genotype or allelic frequencies were observed between RA patients and controls. Conclusions:,RA patients harbour multiple risk factors for statin-induced myopathy. However, the frequency of the rs4149056 genotypes does not differ according to the presence of RA. Despite this, no cases of statin-induced myopathy were observed in this cohort over a period of four years of follow-up. Thus, we conclude that statin use among RA patients is probably safe, but large-scale prospective studies are needed to confirm this. In the meantime, it may be good practice systematically to consider and record myopathy risk factors in these patients. Copyright © 2009 John Wiley & Sons, Ltd [source] Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b genePLANT PATHOLOGY, Issue 1 2009S. Banno Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene (cytb) was characterized. All QoI-resistant isolates had the same mutation (GGT to GCT) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b, which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real-time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI-resistant and QoI-sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI-resistant and QoI-sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild-type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI-sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi-143/144 intron were azoxystrobin-sensitive, suggesting that the QoI-resistant mutation at codon 143 in cytb prevents self-splicing of the Bcbi-143/144 intron, as proposed in some other plant pathogens. [source] Carbamoyl phosphate synthetase I deficiency: molecular genetic findings and prenatal diagnosisPRENATAL DIAGNOSIS, Issue 8 2001Tsutomu Aoshima Abstract We report a Japanese boy who died at Day 28 of life because of severe carbamoyl phosphate synthetase I (CPS1) deficiency that was proven by enzyme assay. By analysis of cDNA and genomic DNA, he was shown to be a compound heterozygote with two point mutations of the CPS1 gene, 840G>C leading to an aberrant splicing and 1123C>T (predicting Q375X). The 840G>C was a mutation described in another Japanese family. Since his parents carried each mutation heterozygously, we performed prenatal diagnosis at 16 weeks of his mother's next gestation by multiplex PCR and melting curve analysis in a single capillary containing two-color fluorescent (LC-Red 640 and LC-Red 705) probes on LightCycler. We analyzed genomic DNA extracted from amniotic cells and found that the fetus was homozygous for the wild-type alleles. At term a healthy girl was born without hyperammonemia. Copyright © 2001 John Wiley & Sons, Ltd. [source] RASSF1A, BLU, NORE1A, PTEN and MGMT Expression and Promoter Methylation in Gliomas and Glioma Cell Lines and Evidence of Deregulated Expression of de novo DNMTsBRAIN PATHOLOGY, Issue 2 2009Aiala Lorente Abstract Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.3), NORE1A (1q32.1) and BLU (3p21.3) have been shown to be downregulated by methylation in cancer, and PTEN (10q23.3) and MGMT (10q26.1) are located in areas commonly deleted in astrocytomas. MGMT methylation predicts a better response and a longer overall survival in patients with glioblastomas treated with temozolomide. We analyzed 53 astrocytoma samples and 10 high-grade glioma cell lines. Gene expression was assessed by RT-PCR. Bisulfite sequencing, MSP and a melting curve analysis-based real-time PCR were performed to detect promoter methylation. Treatments with 5,-aza-2,-deoxicitidine were applied to restore gene expression in cell lines. Ninety-two percent of tumor samples were methylated for RASSF1A, 30%,57% for BLU and 47% for MGMT, suggesting promoter methylation of these genes to be a common event in glioma tumorigenesis. Only 4% of the tumors revealed a methylated promoter for NORE1A. No association between methylation and loss of expression could be established for PTEN. We identified de novo DNMTs overexpression in a subset of tumors which may explain the methylation phenotype of individual gliomas. [source] | |||||||||||||