High-resolution melting: Applications in genetic disorders

Er, Tze‐Kiong; Chang, Jan‐Gowth

doi:10.1016/j.cca.2012.09.012

Cited by 75 publications

(48 citation statements)

References 50 publications

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“…Heterozygotes are easily identified from their melting curves but homozygotes are more difficult to detect. 19) We have identified 2 mutations (p.T309I in KCNQ1 and p.R744fs in KCNH2) and 11 polymorphisms in 5 Taiwanese LQTS patients and the family members of 3 LQTS patients referred for three-gene screening. These mutations and polymorphisms have previously been described.…”

Section: Discussionmentioning

confidence: 99%

Mutation Analysis of KCNQ1, KCNH2 and SCN5A Genes in Taiwanese Long QT Syndrome Patients

et al. 2015

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SummaryLong QT syndrome (LQTS) is a genetic cardiac disease. Gene mutation affects the structure or function of ion channels that are associated with a high risk of sudden death. The goal of this study was to determine the frequency of KCNQ1, KCNH2, and SCN5A mutations in LQTS in a Taiwanese population. Genomic DNA was extracted from peripheral blood samples obtained from 5 patients with LQTS and the family members of 3 LQTS patients. High resolution melting (HRM) analysis and direct DNA sequencing were used to characterize the KCNQ1, KCNH2, and SCN5A genetic variations. HRM analysis was successfully optimized for 14 of the 16 exons of the KCNQ1, 5 of the 15 exons of the KCNH2, and 23 of the 27 exons of the SCN5A. HRM and direct DNA sequencing was applied to the cohort of 5 cases and some of their family. The genetic testing revealed two pathogenic mutations (p.T309I in KCNQ1 and p.R744fs in KCNH2) and all of the mutational frequencies in KCNQ1 and KCNH2 were 20%. In the two patients who carry the pathogenic mutation presenting with recurrent syncope due to ventricular fibrillation, an implantable cardioverter defibrillator was implanted. We also discovered 11 polymorphisms in KCNQ1, 3 in KCNH2, and 5 in SCN5A. Two-fifths of cases (40%) presented with one of the three major LQTS-causing gene mutations. (Int Heart J 2015; 56: 450-453)

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Section: Discussionmentioning

confidence: 99%

Mutation Analysis of KCNQ1, KCNH2 and SCN5A Genes in Taiwanese Long QT Syndrome Patients

et al. 2015

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“…The HRM PCR is a closed-tube and post-PCR method that enables rapid analysis of genetic sequences (Wittwer, 2009;Vossen et al, 2009;Er and Chang, 2012;Wojdacz, 2012). However, few genotypes and markers have been tested and it is unclear whether HRM can be used as an alternative to electrophoresis-based methods for microsatellite detection.…”

Section: Introductionmentioning

confidence: 99%

Y-STR genetic screening by high-resolution melting analysis

et al. 2016

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ABSTRACT. Currently, the widely used automated capillary electrophoresis-based short tandem repeat (STR) genotyping method for genetic screening in forensic practice is laborious, time-consuming, expensive, and technically challenging in some cases. Thus, new molecular-based strategies for conclusively identifying forensically relevant biological evidence are required. Here, we used high-resolution melting analysis (HRM) for Y-chromosome STR genotyping for forensic genetic screening. The reproducibility of the melting profile over dilution, sensitivity, discrimination power, and other factors was preliminarily studied in 10 Y-STR loci. The results showed that HRM-based approaches revealed more genotypes (compared to capillary electrophoresis), showed higher uniformity in replicate tests and diluted samples, and enabled successful detection of DNA at concentrations as low as 0.25 ng. For mixed samples, the melting curve profiles discriminated between mixed samples based on reference samples with high efficiency. The triplex Y-chromosome STR HRM assay was performed and provided a foundation for further studies such as a multiplex HRM assay. The HRM approach is a one-step application and the entire procedure can be completed within 2 h at a low cost. In conclusion, our findings demonstrate that the HRM-based Y-STR assay is a useful screening tool that can be used in forensic practice.

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“…High-resolution melting of PCR products is a simple and popular method for detecting and genotyping sequence variants (1)(2)(3). Scanning for genetic variants by high-resolution melting typically reduces sequencing needs by about 90% (4 ).…”

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confidence: 99%

“…Even when scanning conditions are carefully designed to optimize analytical specificity, polymorphisms that are not of clinical interest are found in about 5% of CFTR [cystic fibrosis transmembrane conductance regulator (ATPbinding cassette sub-family C, member 7)] amplicons (7,8 ) and in 7% of BRCA1 and BRCA2 (breast cancer 2, early onset) amplicons (4,9 ). Although scanning is an inexpensive, simple approach with an analytical sensitivity approaching 100% (1)(2)(3), its advantage over comprehensive sequencing depends strongly on minimizing subsequent analyses.…”

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Symmetric Snapback Primers for Scanning and Genotyping of the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Zhou

Palais

et al. 2013

Clinical Chemistry

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BACKGROUND High-resolution melting of PCR products is an efficient and analytically sensitive method to scan for sequence variation, but detected variants must still be identified. Snapback primer genotyping uses a 5′ primer tail complementary to its own extension product to genotype the resulting hairpin via melting. If the 2 methods were combined to analyze the same PCR product, the residual sequencing burden could be reduced or even eliminated. METHODS The 27 exons and neighboring splice sites of the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene were amplified by the PCR in 39 fragments. Primers included snapback tails for genotyping 7 common variants and the 23 CFTR mutations recommended for screening by the American College of Medical Genetics. After symmetric PCR, the amplicons were analyzed by high-resolution melting to scan for variants. Then, a 5-fold excess of H2O was added to each reaction to produce intramolecular hairpins for snapback genotyping by melting. Each melting step required <10 min. Of the 133 DNA samples analyzed, 51 were from CFTR patient samples or cell lines. RESULTS As expected, the analytical sensitivity of heterozygote detection in blinded studies was 100%. Snapback genotyping reduced the need for sequencing from 7.9% to 0.5% of PCR products; only 1 amplicon every 5 patients required sequencing to identify nonanticipated rare variants. We identified 2 previously unreported variants: c.3945A>G and c.4243–5C>T. CONCLUSIONS CFTR analysis by sequential scanning and genotyping with snapback primers is a good match for targeted clinical genetics, for which high analytical accuracy and rapid turnaround times are important.

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High-resolution melting: Applications in genetic disorders

Cited by 75 publications

References 50 publications

Mutation Analysis of KCNQ1, KCNH2 and SCN5A Genes in Taiwanese Long QT Syndrome Patients

Mutation Analysis of KCNQ1, KCNH2 and SCN5A Genes in Taiwanese Long QT Syndrome Patients

Y-STR genetic screening by high-resolution melting analysis

Symmetric Snapback Primers for Scanning and Genotyping of the Cystic Fibrosis Transmembrane Conductance Regulator Gene

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