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Melting Analysis (melting + analysis)
Selected AbstractsRapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting,HUMAN MUTATION, Issue 10 2008Tomasz K. Wojdacz Abstract Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS. Hum Mutat 0,1,6, 2008. © 2008 Wiley-Liss, Inc. [source] Determination of the mutation spectrum of the EXT1/EXT2 genes in British Caucasian patients with multiple osteochondromas, and exclusion of six candidate genes in EXT negative cases,,HUMAN MUTATION, Issue 11 2006Lorne Lonie Abstract We describe here the spectrum and distribution of mutations in the EXT1 and EXT2 genes in the largest reported British Caucasian multiple osteochondromas (MO) population. Furthermore, we report for the first time the screening of the EXT1 and EXT2 promoters, 5,UTRs, and 3,UTRs, and exclude six potential MO candidate genes in individuals without a detectable mutation within the coding region of EXT1 and EXT2. The coding exons of EXT1 and EXT2 were screened in 72 unrelated probands affected with MO. Forty-six different mutations were identified in 56 probands, of which 29 were novel. Mutation in the EXT1 and EXT2 genes each accounted for 50% of the mutations identified. Of the 72 probands, 42 were of British Caucasian descent, which when added to the 41 British Caucasian families previously reported from our total cohort, gave a total of 83 families. This cohort's proportional frequency for EXT1/EXT2 mutation was 53%/47%. We also validated the technique of high-resolution melting analysis in a blind study using 27 unique EXT1 or EXT2 mutations. This technique was found to be sensitive with a detection rate of 100% regarding heterozygote detection for EXT mutation scanning. Furthermore, this technique has a very high throughput and is very cost-effective. © 2006 Wiley-Liss, Inc. [source] Masking selected sequence variation by incorporating mismatches into melting analysis probes,HUMAN MUTATION, Issue 3 2006Rebecca L. Margraf Abstract Hybridization probe melting analysis can be complicated by the presence of sequence variation (benign polymorphisms or other mutations) near the targeted mutation. We investigated the use of "masking" probes to differentiate alleles with similar probe melting temperatures. Selected sequence variation was masked by incorporating mismatches (deletion, unmatched nucleotide, or universal base) into hybridization probes at the polymorphic location. Such masking probes create a probe/target mismatch with all possible alleles at the selected polymorphic location. Any allele with additional variation at another site is identified by a lower probe melting temperature than alleles that vary only at the masked position. This "masking technique" was applied to RET protooncogene and HPA6 mutation detection using unlabeled hybridization probes, a saturating dsDNA dye, and high-resolution melting analysis. Masking probes clearly distinguished all targeted mutations from polymorphisms when at least 1 base pair (bp) separated the mutation from the masked variation. We were able to mask polymorphisms immediately adjacent to mutations, except in certain cases, such as those involving single-base deletion probes when both adjacent positions had the same polymorphic nucleotides. The masking probes can also localize mutations to specific codons or nucleotide positions. Masking probes can simplify melting analysis of complex regions and eliminate the need for sequencing. Hum Mutat 27(3), 269,278, 2006. © 2006 Wiley-Liss, Inc. [source] Two-state vs. multistate protein unfolding studied by optical melting and hydrogen exchangePROTEIN SCIENCE, Issue 10 2000Leland Mayne Abstract A direct conflict between the stabilization free energy parameters of cytochrome c determined by optical methods and by hydrogen exchange (HX) is quantitatively explained when the partially folded intermediates seen by HX are taken into account. The results support the previous HX measurements of intermediate populations, show how intermediates can elude the standard melting analysis, and illustrate how they confuse the analysis when they are significantly populated within the melting transition region. [source] NFKB1-94ins/del polymorphism is not associated with lung injury after cardiopulmonary bypassANAESTHESIA, Issue 2 2010J. F. Wang Summary Nuclear factor (NF)-,B (NFKB1)-94ins/del is an important polymorphism that affects promoter activity of the NFKB1 gene and is potentially associated with several inflammatory diseases. We investigated the association of this polymorphism with lung injury after cardiac surgery and cardiopulmonary bypass in a prospective cohort study of 283 patients. Genotyping was performed by high resolution melting analysis; analysis indicated no association of NFKB1 with postoperative lung injury (p = 0.064). Relative risks of the del allele and the del/del genotype were 1.34 (95% CI 1.02,1.75) and 1.74 (95% CI 1.00,3.05) respectively. Logistic regression analysis (with factors including age, peripheral vascular disease and surgical duration as risk factors of lung injury after cardiac surgery with cardiopulmonary bypass) also failed to confirm that the NFKB1 genotype is influential for lung injury (p = 0.113). We conclude that, contrary to some other evidence, the NFKB1-94ins/del polymorphism is not associated with lung injury after cardiac surgery with cardiopulmonary bypass. [source] Epidermal growth factor receptor mutation status and clinicopathological features of combined small cell carcinoma with adenocarcinoma of the lungCANCER SCIENCE, Issue 11 2007Tomoya Fukui In lung cancer, somatic mutations of epidermal growth factor receptor (EGFR) are concentrated in exons 18,21, especially in adenocarcinoma (Ad), but these mutations have rarely been reported in small cell lung carcinoma (SCLC). Combined SCLC is rare, and the EGFR mutation status and its relationship to the clinicopathological features of this tumor type have not yet been elucidated. We retrospectively studied six patients with combined SCLC with Ad components among 64 consecutive patients who underwent resection of SCLC. The clinicopathological features of each patient were reviewed, especially for the distribution pattern of the Ad component and lymph node metastases. EGFR mutations were screened by high-resolution melting analysis in each case, and were confirmed by sequencing of each mutation in the microdissected SCLC or Ad components. Regarding EGFR, no specific mutation was detected in five of the six patients, whereas one female patient who had never smoked had a missense mutation. In this case, both the SCLC and Ad components shared the same mutation in exon 21 (L858R). We identified a patient with combined SCLC with Ad sharing an identical EGFR mutation in both the SCLC and Ad components. In addition to the clinicopathological characteristics of this rare histological type of lung cancer, these findings provide useful information for better understanding the biology, natural history and clinical management of SCLC. (Cancer Sci 2007; 98: 1714,1719) [source] 2-Phenanthrenyl,DNA: Synthesis, Pairing, and Fluorescence PropertiesCHEMISTRY - A EUROPEAN JOURNAL, Issue 3 2009Nikolay Abstract Three 2,-phenanthrenyl- C -deoxyribonucleosides with donor (phenNH2), acceptor (phenNO2), or no (phenH) substitution on the phenanthrenyl core were synthesized and incorporated into oligodeoxyribonucleotides. Duplexes containing either one or three consecutive phenR residues, which were located opposite each other, were formed. Within these residues, the phenR residues are expected to recognize each other through interstrand stacking interactions, in much the same way as described previously for biphenyl DNA. The thermal, thermodynamic, and fluorescence properties of such duplexes were determined by UV melting analysis and fluorescence spectroscopy. Depending on the nature of the substituent, the thermal stability of single-modified duplexes can vary between ,2.7 to +11.3,°C in Tm and that of triple-modified duplexes from +7.8 to +11.1,°C. Van,t Hoff analysis suggested that the observed higher thermodynamic stability in phenH- and phenNO2 -containing duplexes is of enthalpic origin. A single phenH or phenNO2 residue in a bulge position also stabilizes a corresponding duplex. If a phenNO2 residue is placed in a bulge position next to a base mismatch this can lead, in a sequence-dependent manner, to duplex destabilization. The phenNO2 residue was found to be a highly efficient (10,100-fold) quencher of phenH and phenNH2 fluorescence if placed in the opposite position to the fluorophores. When phenH and phenNH2 residues were placed opposite each other, efficient quenching of phenH and enhancement of phenNH2 fluorescence was found, which is an indicator for electron- or energy-transfer processes between the aromatic units. 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