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doi: 10.1002/humu.20951pmid: 19479960
Recent advances in fluorescent dyes, methods, instruments and software for DNA melting analysis have created versatile new tools for variant scanning and genotyping. High resolution melting analysis (HRM or HRMA) is faster, simpler, and less expensive than alternative approaches requiring separations or labeled probes. With the addition of a saturating dye before PCR followed by rapid melting analysis of the PCR products, the sensitivity of heterozygote scanning approaches 100%. Specificity can be increased by identifying common polymorphisms with small amplicon melting, unlabeled probes or snapback primers to decrease the sequencing burden. However, some homozygotes require mixing for identification. Furthermore, different heterozygotes may produce melting curves so similar to each other that, although they clearly vary from homozygous variants, they are not differentiated from each other. Nevertheless, the experimental return for minimal effort is great. This focus issue of Human Mutation includes a concise, timely review on high resolution melting, a comparison to denaturing gradient gel electrophoresis, integration with qPCR for copy number assessment, combined amplicon scanning and unlabeled probe genotyping from a single melting curve, and applications to the mitochondrial genome and to BRCA1. Hum Mutat 30, 857–859, 2009. © 2009 Wiley‐Liss, Inc.
Vossen, Rolf H.A.M.; Aten, Emmelien; Roos, Anja; den Dunnen, Johan T.
doi: 10.1002/humu.21019pmid: 19418555
Transition of the double‐stranded DNA molecule to its two single strands, DNA denaturation or melting, has been used for many years to study DNA structure and composition. Recent technological advances have improved the potential of this technology, especially to detect variants in the DNA sequence. Sensitivity and specificity were increased significantly by the development of so‐called saturating DNA dyes and by improvements in the instrumentation to measure the melting behavior (improved temperature precision combined with increased measurements per time unit and drop in temperature). Melt analysis using these new instruments has been designated high‐resolution melting curve analysis (HRM or HRMA). Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity, HRMA is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we will briefly discuss the latest developments in HRMA and review in particular other applications that have thus far received less attention, including presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants and mosaicism), an alternative to gel‐electrophoresis and clone characterization. Together, these diverse applications make HRMA a multipurpose technology and a standard tool that should be present in any laboratory studying nucleic acids. Hum Mutat 30:1–7, 2009. © 2009 Wiley‐Liss, Inc.
Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette
doi: 10.1002/humu.20947pmid:
Tindall, Elizabeth A.; Petersen, Desiree C.; Woodbridge, Paula; Schipany, Katharina; Hayes, Vanessa M.
doi: 10.1002/humu.20919pmid: 19280649
Mutation detection has, until recently, relied heavily on the use of gel‐based methods that can be both time consuming and difficult to design. Nongel‐based systems are therefore important to increase simplicity and improve turn around time without compromising assay sensitivity and accuracy, especially in the diagnostic/clinical setting. In this study, we assessed the latest of the nongel‐based methods, namely high‐resolution melt (HRM) curve analysis. HRM is a closed‐tube method that incorporates a saturating dye during DNA amplification followed by a monitoring of the change in fluorescence as the DNA duplex is denatured by an increasing temperature. We assessed 10 amplicons derived from eight genes, namely SERPINA1, CXCR7, MBL, VDR, NKX3A, NPY, TP53, and HRAS using two platforms, the LightScanner® System using LC Green® PLUS DNA binding dye (Idaho Technology, Salt Lake City, UT, USA) and the LightCycler® 480 using the HRM Master dye (Roche Diagnostics, Indianapolis, IN, USA). DNA variants (mutations or polymorphims) were previously identified using denaturing gradient gel electrophoresis (DGGE) a method, similarly to HRM, based upon the different melting properties of double‐stranded DNA. Fragments were selected based on variant and fragment complexity. This included the presence of multiple sequence variants, variants in alternate orientations, and single or multiple variants (constitutional or somatic) in GC‐rich fragments. We demonstrate current limitations of the HRM method for the analysis of complex DNA regions and call for caution when using HRM as the sole method to make a clinical diagnosis based on genetic analysis. Hum Mutat 30, 1–8, 2009 © 2009 Wiley‐Liss, Inc.
Nguyen‐Dumont, Tú; Calvez‐Kelm, Florence Le; Forey, Nathalie; McKay‐Chopin, Sandrine; Garritano, Sonia; Gioia‐Patricola, Lydie; De Silva, Deepika; Weigel, Ron; Sangrajrang, Suleeporn; Lesueur, Fabienne; Tavtigian, Sean V.
doi:
Dobrowolski, Steven F.; Gray, Jesse; Miller, Trent; Sears, Mitch
doi: 10.1002/humu.21003pmid: 19370763
Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. High‐resolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301–658 bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384‐well plate or two 96‐well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke‐like episodes (MELAS) mutation m.3243A>G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A>G, and m.1555A>G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon‐based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1 hr. Hum Mutat 30,1–8, 2009. © 2009 Wiley‐Liss, Inc.
van der Stoep, Nienke; van Paridon, Chantal D.M.; Janssens, Tom; Krenkova, Petra; Stambergova, Alexandra; Macek, Milan; Matthijs, Gert; Bakker, Egbert
doi: 10.1002/humu.21004pmid: 19370767
Genetic analysis of BRCA1 by sequencing is often preceded by a scanning method like denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) or DHPLC. High‐resolution melting curve (HRM) analysis is a promising and economical method for high‐throughput mutation scanning. The EuroGentest network (www.eurogentest.org) aims to assist with the introduction of novel technologies in the diagnostic setting. Therefore, we have performed a thorough and high‐standard interlaboratory evaluation and validation of HRM, in collaboration with Idaho Technology, the manufacturer of the LightScannerTM (LS). Through this detailed study of 170 variants, we have generated guidelines for easy setup and implementation of HRM as a scanning technique for new genes, which are adaptable to the quality system of an individual diagnostic laboratory. This validation study includes the description of a BRCA1‐specific mutation screening test using the 96‐well LS. This assay comprises 40 amplicons and was evaluated using a statistically significant elaborate panel of variants and control DNA samples. All heterozygous variants were detected. Moreover, genotype analysis for nine common polymorphisms created a fast screening and detection method for these frequently occurring nonpathogenic variants. A blind study using a total of 28 patient‐derived DNA samples resulted also in 100% detection and showed an average specificity of 98%, indicating a low incidence of false positives (FPs). Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.
Showing 1 to 10 of 25 Articles
Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called “quantitative PCR (qPCR) high‐resolution melting (HRM) curve analysis (qPCR‐HRM),” which combines qPCR and HRM to obtain a rapid and cost‐effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy‐six patients were fully scanned in replicate, including 14 wild‐type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array–comparative genomic hybridization (array‐CGH). All point mutations and rearrangements detected by denaturing high‐performance liquid chromatography (dHPLC)+multiplex ligation‐dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array‐CGH. One variant was detected with qPCR HRM in a wild‐type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T‐stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided. Hum Mutat 0, 1–9, 2009. © 2009 Wiley‐Liss, Inc.
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.