scholarly journals New sequence variants in BRCA1 and BRCA2 genes detected by high-resolution melting analysis in an elderly healthy female population in Croatia

2008 ◽  
Vol 46 (10) ◽  
Author(s):  
Mirela Levacic Cvok ◽  
Maja Cretnik ◽  
Vesna Musani ◽  
Petar Ozretic ◽  
Sonja Levanat
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Sequence Variants ◽  
Brca1 And Brca2 ◽  
Female Population ◽  
Healthy Female ◽  
Brca1 And Brca2 Genes ◽  
Melting Analysis

scholarly journals Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

2003 ◽  
Vol 49 (3) ◽  
pp. 396-406 ◽  
Author(s):  
Cameron N Gundry ◽  
Joshua G Vandersteen ◽  
Gudrun H Reed ◽  
Robert J Pryor ◽  
Jian Chen ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Rapid Heating ◽  
Melting Curve ◽  
Pcr Primers ◽  
High Resolution Melting Analysis ◽  
Melting Transition ◽  
Sequence Variant ◽  
Sequence Variants ◽  
Melting Analysis

Abstract Background: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (Tm). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ∼1 min and no sample processing was needed after PCR. Results: Polymorphisms in the HTR2A (T102C), β-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 °C/s) of denatured products, followed by rapid heating during melting analysis (0.2–0.4 °C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in Tm by <0.2 °C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5′ tail of identical sequence was added to one of the two unlabeled primers. Conclusion: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.

scholarly journals Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting Platforms

2008 ◽  
Vol 54 (6) ◽  
pp. 982-989 ◽  
Author(s):  
Kim De Leeneer ◽  
Ilse Coene ◽  
Bruce Poppe ◽  
Anne De Paepe ◽  
Kathleen Claes
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Turnaround Time ◽  
High Resolution Melting Analysis ◽  
Coding Region ◽  
Brca1 And Brca2 ◽  
Diagnostic Laboratory ◽  
Sensitivity Setting ◽  
Melting Analysis

Abstract Background: High-resolution melting is an emerging technique for detection of nucleic acid sequence variations. Developments in instrumentation and saturating intercalating dyes have made accurate high-resolution melting analysis possible and created opportunities to use this technology in diagnostic settings. We evaluated 2 high-resolution melting instruments for screening BRCA1 and BRCA2 mutations. Methods: To cover the complete coding region and splice sites, we designed 112 PCR amplicons (136–435 bp), amplifiable with a single PCR program. LCGreen® Plus was used as the intercalating dye. High-resolution melting analysis was performed on the 96-well Lightscanner™ (Idaho Technology Inc.) and the 96-well LightCycler® 480 (Roche) instruments. We evaluated sensitivity by analyzing 212 positive controls scattered over almost all amplicons and specificity by blind screening of 22 patients for BRCA1 and BRCA2. In total, we scanned 3521 fragments. Results: All 212 known heterozygous sequence variants were detected on the Lightscanner by analysis on normal sensitivity setting. On the LightCycler 480, the standard instrument sensitivity setting of 0.3 had to be increased to 0.7 to detect all variants, decreasing the specificity to 95.9% (vs 98.7% for the Lightscanner). Conclusions: Previously, we screened BRCA1/2 by direct sequencing of the large exon 11 and denaturing gel gradient electrophoresis (DGGE) for all other coding exons. Since the introduction of high-resolution melting, our turnaround time has been one third of that with direct sequencing and DGGE, as post-PCR handling is no longer required and the software allows fast analyses. High-resolution melting is a rapid, cost-efficient, sensitive method simple enough to be readily implemented in a diagnostic laboratory.

High-resolution melting analysis for rapid screening of BRCA1 and BRCA2 Spanish mutations

2008 ◽  
Vol 115 (2) ◽  
pp. 405-414 ◽  
Author(s):  
Inmaculada de Juan ◽  
Eva Esteban ◽  
Sarai Palanca ◽  
Eva Barragán ◽  
Pascual Bolufer
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Rapid Screening ◽  
Brca1 And Brca2 ◽  
Melting Analysis

scholarly journals Parental mosaicism in Marfan and Ehlers–Danlos syndromes and related disorders

2021 ◽  
Author(s):  
Bertrand Chesneau ◽  
Aurélie Plancke ◽  
Guillaume Rolland ◽  
Nicolas Chassaing ◽  
Christine Coubes ◽  
...  
Keyword(s):  
High Resolution ◽  
Marfan Syndrome ◽  
High Resolution Melting ◽  
De Novo ◽  
Direct Sequencing ◽  
Causal Variant ◽  
Connective Tissue Disorder ◽  
High Resolution Melting Analysis ◽  
Aortic Diseases ◽  
Melting Analysis

AbstractMarfan syndrome (MFS) is a heritable connective tissue disorder (HCTD) caused by pathogenic variants in FBN1 that frequently occur de novo. Although individuals with somatogonadal mosaicisms have been reported with respect to MFS and other HCTD, the overall frequency of parental mosaicism in this pathology is unknown. In an attempt to estimate this frequency, we reviewed all the 333 patients with a disease-causing variant in FBN1. We then used direct sequencing, combined with High Resolution Melting Analysis, to detect mosaicism in their parents, complemented by NGS when a mosaicism was objectivized. We found that (1) the number of apparently de novo events is much higher than the classically admitted number (around 50% of patients and not 25% as expected for FBN1) and (2) around 5% of the FBN1 disease-causing variants were not actually de novo as anticipated, but inherited in a context of somatogonadal mosaicisms revealed in parents from three families. High Resolution Melting Analysis and NGS were more efficient at detecting and evaluating the level of mosaicism compared to direct Sanger sequencing. We also investigated individuals with a causal variant in another gene identified through our “aortic diseases genes” NGS panel and report, for the first time, on an individual with a somatogonadal mosaicism in COL5A1. Our study shows that parental mosaicism is not that rare in Marfan syndrome and should be investigated with appropriate methods given its implications in patient’s management.

scholarly journals Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis

2016 ◽  
Vol 54 (7) ◽  
pp. 714-724 ◽  
Author(s):  
Matej Bezdicek ◽  
Martina Lengerova ◽  
Dita Ricna ◽  
Barbora Weinbergerova ◽  
Iva Kocmanova ◽  
...  
Keyword(s):  
High Resolution ◽  
Bronchoalveolar Lavage ◽  
Rapid Detection ◽  
Fungal Pathogens ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Melting Analysis ◽  
Panfungal Pcr

High-resolution melting analysis for detection of MYH9 mutations

Platelets ◽  
2008 ◽  
Vol 19 (6) ◽  
pp. 471-475 ◽  
Author(s):  
Dana Provaznikova ◽  
Tereza Kumstyrova ◽  
Roman Kotlin ◽  
Peter Salaj ◽  
Vaclav Matoska ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Melting Analysis
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