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Journal articles on the topic 'High resolution melting analysis'

Author: Grafiati

Published: 4 June 2021

Last updated: 10 March 2023

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1

Carillo, Serge, Laurent Henry, Eric Lippert, François Girodon, Isabelle Guiraud, Céline Richard, Frédérique Dubois Galopin, et al. "Nested High-Resolution Melting Curve Analysis." Journal of Molecular Diagnostics 13, no. 3 (May 2011): 263–70. http://dx.doi.org/10.1016/j.jmoldx.2010.12.002.

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Ro, Na Young, On Sook Hur, Ho Cheol Ko, Sang Gyu Kim, Ju Hee Rhee, Jae-Gyun Gwag, Jin-Kyung Kwon, and Byoung-Cheorl Kang. "Evaluation of Resistance in Pepper Germplasm to Cucumber mosaic virus by High Resolution Melting Analysis." Research in Plant Disease 18, no. 4 (December 30, 2012): 290–97. http://dx.doi.org/10.5423/rpd.2012.18.4.290.

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3

Erali, Maria, and Carl T. Wittwer. "High resolution melting analysis for gene scanning." Methods 50, no. 4 (April 2010): 250–61. http://dx.doi.org/10.1016/j.ymeth.2010.01.013.

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4

Tong, S. Y. C., and P. M. Giffard. "Microbiological Applications of High-Resolution Melting Analysis." Journal of Clinical Microbiology 50, no. 11 (August 8, 2012): 3418–21. http://dx.doi.org/10.1128/jcm.01709-12.

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5

Zambounis, Antonios, Eleni Stefanidou, Panagiotis Madesis, Jovana Hrustić, Milica Mihajlović, and Brankica Tanović. "Genotypic differentiation of Monilinia spp. populations in Serbia using a high-resolution melting (HRM) analysis." Plant Protection Science 57, No. 1 (December 3, 2020): 38–46. http://dx.doi.org/10.17221/35/2020-pps.

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Monilinia laxa, Monilinia fructicola and Monilinia fructigena are the three main causal agents of brown rot, which is one of the most important diseases of stone fruits in pre- and postharvest conditions. Nowadays, the need for the precise genotyping of these Monilinia species in terms of the genetic diversity of their populations or differences in their pathogenicity and host range is a prerequisite for any efficient disease management. In our study, the genetic structure of Monilinia populations in Serbia from three geographically distinct regions was investigated employing <br /> a high-resolution melting (HRM) analysis which is a sensitive and rapid molecular approach in fungal genotyping and diagnostics. Using species-specific primer pairs genotype-specific HRM melting curve profiles were generated allowing to efficiently decipher the genetic diversity of the Monilinia populations. The Monilinia genotypes could be easily distinguished according to their melting curves. The isolates from the northern region were assigned to distinct genotypes and grouped rather independently compared to the isolates of the other two regions among all three tested Monilinia spp. M. fructicola and M. fructigena showed a higher genetic diversity among their populations (44%) compared with the genetic diversity among the M. laxa populations (7%). In contrast, the genetic variance within the pathogen populations was higher in the case of M. laxa (93%). Our data revealed an absence of host specificity in the Monilinia spp. populations.

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Zumaraga, Mark Pretzel, Marietta Rodriguez, Vanessa Joy Timoteo, and Celeste Tanchoco. "Method Validation of a High Resolution Melting Analysis of a Candidate Genetic Marker of Hypertension." Journal of the ASEAN Federation of Endocrine Societies 30, no. 1 (May 31, 2015): 18–24. http://dx.doi.org/10.15605/jafes.030.01.01.

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7

Wittwer, Carl T., Gudrun H. Reed, Cameron N. Gundry, Joshua G. Vandersteen, and Robert J. Pryor. "High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen." Clinical Chemistry 49, no. 6 (June 1, 2003): 853–60. http://dx.doi.org/10.1373/49.6.853.

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Abstract Background: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396–406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), β-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. Results: The six common β-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1–2 min, amplification and analysis were achieved in 10–20 min with rapid cycling conditions. Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.

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8

Simko, Ivan. "High-Resolution DNA Melting Analysis in Plant Research." Trends in Plant Science 21, no. 6 (June 2016): 528–37. http://dx.doi.org/10.1016/j.tplants.2016.01.004.

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9

Mader, Eduard, Joana Ruzicka, Corinna Schmiderer, and Johannes Novak. "Quantitative high-resolution melting analysis for detecting adulterations." Analytical Biochemistry 409, no. 1 (February 2011): 153–55. http://dx.doi.org/10.1016/j.ab.2010.10.009.

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10

Janavicius, Ramunas, Dovile Matiukaite, Arturas Jakubauskas, and Laimonas Griskevicius. "Microsatellite Instability Detection by High-Resolution Melting Analysis." Clinical Chemistry 56, no. 11 (November 1, 2010): 1750–57. http://dx.doi.org/10.1373/clinchem.2010.150680.

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BACKGROUND Microsatellite instability (MSI) is an important marker for screening for hereditary nonpolyposis colorectal cancer (Lynch syndrome) as well as a prognostic and predictive marker for sporadic colorectal cancer (CRC). The mononucleotide microsatellite marker panel is a well-established and superior alternative to the traditional Bethesda MSI analysis panel, and does not require testing for corresponding normal DNA. The most common MSI detection techniques—fluorescent capillary electrophoresis and denaturing HPLC (DHPLC)—both have advantages and drawbacks. A new high-resolution melting (HRM) analysis method enables rapid identification of heteroduplexes in amplicons by their lower thermal stability, a technique that overcomes the main shortcomings of capillary electrophoresis and DHPLC. METHODS We investigated the straightforward application of HRM for the detection of MSI in 70 archival CRC samples. HRM analysis for 2 MSI markers (BAT25 and BAT26) was evaluated, and 2 different HRM-enabled instruments were compared—the LightCycler® 480 (Roche Diagnostics) and the LightScannerTM (Idaho Technology). We also determined the analytical sensitivity and specificity of the HRM assay on both instruments using 11 known MSI-positive and 54 microsatellite-stable CRC samples. RESULTS All MSI-positive samples were detected on both instruments (100% analytical sensitivity). The LightScanner performed better for analytical specificity, giving a combined specificity value of 99.1% compared with 92.3% on the LightCycler 480. CONCLUSIONS We expanded the application of the HRM analysis method as an effective MSI detection technique for clinical samples.

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11

Wittwer, Carl T. "High-resolution DNA melting analysis: advancements and limitations." Human Mutation 30, no. 6 (June 2009): 857–59. http://dx.doi.org/10.1002/humu.20951.

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Antonios, Zambounis, Samaras Anastasios, Xanthopoulou Aliki, Osathanunkul Maslin, Schena Leonardo, Tsaftaris Athanasios, and Madesis Panagiotis. "Identification of Phytophthora species by a high resolution melting analysis: an innovative tool for rapid differentiation." Plant Protection Science 52, No. 3 (May 26, 2016): 176–81. http://dx.doi.org/10.17221/179/2015-pps.

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13

Laurie, Andrew D., Mark P. Smith, and Peter M. George. "Detection of Factor VIII Gene Mutations by High-Resolution Melting Analysis." Clinical Chemistry 53, no. 12 (December 1, 2007): 2211–14. http://dx.doi.org/10.1373/clinchem.2007.093781.

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Abstract Background: Single base-pair substitution mutations in the gene for coagulation factor VIII, procoagulant component (hemophilia A) (F8) account for approximately 50% of severe cases of hemophilia A (HA), and almost all moderate or mild cases. Because F8 is a large gene, mutation screening using denaturing HPLC or DNA sequencing is time-consuming and expensive. Methods: We evaluated high-resolution melting analysis as an option for screening for F8 gene mutations. The melting curves of amplicons heterozygous for known F8 gene mutations were compared with melting curves of the corresponding normal amplicons to assess whether melting analysis could detect these variants. We examined 2 platforms, the Roche LightCycler 480 (LC480) and the Idaho Technology LightScanner. Results: On both instruments, 18 (90%) of the 20 F8 gene variants we examined were resolved by melting analysis. For the other 2 mutations, the melting curves of the heterozygous amplicons were similar to the corresponding normal amplicons, suggesting these variants may not be detected by this approach in a mutation-scanning screen. Conclusion: High-resolution melting analysis is an appealing technology for F8 gene screening. It is rapid and quickly identifies mutations in the majority of HA patients; samples in which no mutation is detected require further testing by DNA sequencing. The LC480 and LightScanner platforms performed similarly.

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Li, Huaizhong, Ruiting Lan, Niancai Peng, Jing Sun, and Yong Zhu. "High resolution melting curve analysis with MATLAB-based program." Measurement 90 (August 2016): 178–86. http://dx.doi.org/10.1016/j.measurement.2016.04.057.

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15

Schmiderer, Corinna, Eduard Mader, and Johannes Novak. "DNA-Based Identification ofHelleborus nigerby High-Resolution Melting Analysis." Planta Medica 76, no. 16 (May 7, 2010): 1934–37. http://dx.doi.org/10.1055/s-0030-1249908.

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16

Provaznikova, Dana, Tereza Kumstyrova, Roman Kotlin, Peter Salaj, Vaclav Matoska, Ingrid Hrachovinova, and Simon Rittich. "High-resolution melting analysis for detection of MYH9 mutations." Platelets 19, no. 6 (January 2008): 471–75. http://dx.doi.org/10.1080/09537100802140013.

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17

Slany, Michal, Martina Vanerkova, Eva Nemcova, Barbora Zaloudikova, Filip Ruzicka, and Tomas Freiberger. "Differentiation of Staphylococcus spp. by high-resolution melting analysis." Canadian Journal of Microbiology 56, no. 12 (December 2010): 1040–49. http://dx.doi.org/10.1139/w10-091.

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High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains ( Staphylococcus aureus , Staphylococcus capitis , Staphylococcus caprae , Staphylococcus epidermidis , Staphylococcus haemolyticus , Staphylococcus hominis , Staphylococcus intermedius , Staphylococcus saprophyticus , Staphylococcus sciuri , Staphylococcus simulans , Staphylococcus warneri , and Staphylococcus xylosus ) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

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Rojo, Daniela, Manuel Zapata, Alejandro Maureira, Ricardo Guiñez, Cristian Wulff-Zottele, and Mariella Rivas. "High-resolution melting analysis for identification of microalgae species." Journal of Applied Phycology 32, no. 6 (September 11, 2020): 3901–11. http://dx.doi.org/10.1007/s10811-020-02240-y.

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19

Cho, Michael H., Dawn Ciulla, Barbara J. Klanderman, Benjamin A. Raby, and Edwin K. Silverman. "High-Resolution Melting Curve Analysis of Genomic and Whole-Genome Amplified DNA." Clinical Chemistry 54, no. 12 (December 1, 2008): 2055–58. http://dx.doi.org/10.1373/clinchem.2008.109744.

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Abstract Background: High-resolution melting curve analysis is an accurate method for mutation detection in genomic DNA. Few studies have compared the performance of high-resolution DNA melting curve analysis (HRM) in genomic and whole-genome amplified (WGA) DNA. Methods: In 39 paired genomic and WGA samples, 23 amplicons from 9 genes were PCR amplified and analyzed by high-resolution melting curve analysis using the 96-well LightScanner (Idaho Technology). We used genotyping and bidirectional resequencing to verify melting curve results. Results: Melting patterns were concordant between the genomic and WGA samples in 823 of 863 (95%) analyzed sample pairs. Of the discordant patterns, there was an overrepresentation of alternate melting curve patterns in the WGA samples, suggesting the presence of a mutation (false positives). Targeted resequencing in 135 genomic and 136 WGA samples revealed 43 single nucleotide polymorphisms (SNPs). All SNPs detected in genomic samples were also detected in WGA. Additional genotyping and sequencing allowed the classification of 628 genomic and 614 WGA amplicon samples. Heterozygous variants were identified by non–wild-type melting pattern in 98% of genomic and 97% of WGA samples (P = 0.11). Wild types were correctly classified in 99% of genomic and 91% of WGA samples (P < 0.001). Conclusions: In WGA DNA, high-resolution DNA melting curve analysis is a sensitive tool for SNP discovery through detection of heterozygote variants; however, it may misclassify a greater number of wild-type samples.

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Knopkiewicz, M., M. Gawłowska, and W. Święcicki. "The application of high resolution melting in the analysis of simple sequence repeat and single nucleotide polymorphism markers in a pea (Pisum sativum L.) population." Czech Journal of Genetics and Plant Breeding 50, No. 2 (June 12, 2014): 151–56. http://dx.doi.org/10.17221/113/2013-cjgpb.

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The aim of this study was to verify the high resolution melting (HRM) method in the analysis of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in pea (Pisum sativum L.). A recombinant inbred line population, Carneval × MP1401, was tested for three SNP and 103 SSR markers. HRM analysis was conducted on a LightScanner 96 instrument with LC Green dye. The melting curve shape permitted two polymorphic genotypes to be distinguished. The results were confirmed by gel electrophoresis. Three SSR markers were sequenced and analysed by the melting prediction software. The results confirmed the presence of one polymerase chain reaction (PCR) product with two melting domains. Sequence tagged site (STS) markers produced specific products: Psat_EST_00189_01_1 (300 bp), Pis_GEN_18_2_1 (400 bp), Pis_GEN_7_1-2_1 (600 bp). Amplicons contained one, four and seven single nucleotide polymorphisms, respectively. Melting curve differences enabled the population genotyping except for Psat_EST_00189_01_1 where resolution was too low. Primers for Psat_EST_00189_01_1 were redesigned to obtain a shorter (100 bp) PCR product which increased the resolution. The number of SNPs and amplicon length are crucial for HRM resolution. The HRM method is fast and has a high throughput. The melting analysis of 96 samples takes less than 10 min. Agarose gel analysis confirmed the reliability of HRM, which eliminates laborious post-PCR analysis.

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Vandersteen, Joshua G., Pinar Bayrak-Toydemir, Robert A. Palais, and Carl T. Wittwer. "Identifying Common Genetic Variants by High-Resolution Melting." Clinical Chemistry 53, no. 7 (July 1, 2007): 1191–98. http://dx.doi.org/10.1373/clinchem.2007.085407.

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Abstract Background: Heteroduplex scanning techniques usually detect all heterozygotes, including common variants not of clinical interest. Methods: We conducted high-resolution melting analysis on the 24 exons of the ACVRL1 and ENG genes implicated in hereditary hemorrhagic telangiectasia (HHT). DNA in samples from 13 controls and 19 patients was PCR amplified in the presence of LCGreen® I, and all 768 exons melted in an HR-1® instrument. We used 10 wild-type controls to identify common variants, and the remaining samples were blinded, amplified, and analyzed by melting curve normalization and overlay. Unlabeled probes characterized the sequence of common variants. Results: Eleven common variants were associated with 8 of the 24 HHT exons, and 96% of normal samples contained at least 1 variant. As a result, the positive predictive value (PPV) of a heterozygous exon was low (31%), even in a population of predominantly HHT patients. However, all common variants produced unique amplicon melting curves that, when considered and eliminated, resulted in a PPV of 100%. In our blinded study, 3 of 19 heterozygous disease-causing variants were missed; however, 2 were clerical errors, and the remaining false negative would have been identified by difference analysis. Conclusions: High-resolution melting analysis is a highly accurate heteroduplex scanning technique. With many exons, however, use of single-sample instruments may lead to clerical errors, and routine use of difference analysis is recommended. Common variants can be identified by their melting curve profiles and genotyped with unlabeled probes, greatly reducing the false-positive results common with scanning techniques.

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Shestakova, Anna, Felipe Lorenzo, Tsewang Tashi, Lucie Lanikova, Carl T. Wittwer, and Josef T. Prchal. "Tibetan PHD2D4E High Altitude Adapted Gene Can be Rapidly Detected By High Resolution Melting Assay." Blood 124, no. 21 (December 6, 2014): 4875. http://dx.doi.org/10.1182/blood.v124.21.4875.4875.

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Abstract High altitude is accompanied by hypoxia. Acute and chronic hypoxia induces a number of compensatory physiological responses mediated by hypoxia-inducible factors (HIFs) that regulate erythropoiesis, iron and energy metabolism, and other essential organismal responses. Excessive HIF responses occurring at high altitude may be accompanied by morbidity (polycythemia and pulmonary hypertension) or mortality (brain and pulmonary edema). HIFs are down regulated by two principal factors, i.e. prolyl hydroxylases (PHDs) and von Hippel Lindau proteins (VHL). Tibetans have lived at 3,000-5,000 meters for approximately 20,000 years and have acquired a number of beneficial genetic adaptations which appear to prevent negative responses to hypoxia at high-altitude. Deciphering these genetic changes is crucial to improve our understanding of the underlying hypoxia-mediated response mechanisms and to develop targeted therapies. We recently identified the first Tibetan-specific mutation, PHD2D4E, caused by a missense mutation (rs186996510) in EGLN1. PHD2D4E has an allelic frequency of ~85% in Tibetans and a low Km for oxygen, accounting for the protection of Tibetans from high-altitude polycythemia. Other effects of PHD2D4E on HIF-regulated pathophysiology remain to be delineated. A 77% GC-rich area surrounds rs186996510, resulting in a low success rate of detecting the mutation by Sanger sequencing or next-generation sequencing. PHD2D4E was unreported in published whole-genome analyses of Tibetans (Xin Yi et. al. Science 2010). Here we describe a high-resolution melting assay of a small PCR product for targeted genotyping of rs186996510. The single base-pair change (G to C) is visualized by melting small amplicons in the presence of a fluorescent DNA-binding dye. Heterozygotes are differentiated from homozygous genotypes by a pronounced change in the shape of the melting curve caused by the formation of heteroduplexes. However, wild type and homozygous variants are difficult to distinguish by melting alone, and require an additional step of a second melting analysis after mixing with known wild type DNA. Upon melting these mixtures, homozygotes appear as heterozygous melting curves, while wild type genotypes will remain wild type (Figure 1). We developed and validated a high resolution melting assay for rapid genotyping of PHD2D4E suitable for population and disease association studies. In our ongoing analyses, we genotyped DNA from over 300 Tibetans residing at sea level, 1300 meters, 1730-2300 meters and 4320 meters, and are correlating the allelic frequency of PHD2D4E with hematocrit levels. The high resolution melting assay for genotyping PHD2D4E is a simple, accurate, rapid, and inexpensive approach to identify SNP-targeted mutations, especially suitable for a large number of samples such as needed for population studies, without the expense and time required for sequencing studies. Figure 1. High resolution melting analysis of rs186996510 using a 48-base a pair PCR product amplified with primers Forward 5Õ AACGCTCTCACGCCGCCATGGCCAATGA 3Õ and Reverse 5Õ GCCGGGCCCGCCGCT 3Õ. Rapid-cycle PCR amplification and melting analysis were performed in a LS32 real-time instrument. Amplicons from homozygous, heterozygous and wild-type genotypes, and a mixture of wild-type and homozygous products were melted in the presence of a saturating DNA dye (LCGreen). High resolution melting curves and derivative plot are shown. Heterozygotes, or mixed wild type and homozygous variant produce a large change in the shape of the melting curve (red) in comparison to wild-type and homozygous variant (black). Figure 1. High resolution melting analysis of rs186996510 using a 48-base a pair PCR product amplified with primers Forward 5Õ AACGCTCTCACGCCGCCATGGCCAATGA 3Õ and Reverse 5Õ GCCGGGCCCGCCGCT 3Õ. Rapid-cycle PCR amplification and melting analysis were performed in a LS32 real-time instrument. Amplicons from homozygous, heterozygous and wild-type genotypes, and a mixture of wild-type and homozygous products were melted in the presence of a saturating DNA dye (LCGreen). High resolution melting curves and derivative plot are shown. Heterozygotes, or mixed wild type and homozygous variant produce a large change in the shape of the melting curve (red) in comparison to wild-type and homozygous variant (black). Disclosures Wittwer: BioFire Diagnostics: Aspects of melting analysis Patents & Royalties, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Margraf, Rebecca L., Rong Mao, W. Edward Highsmith, Leonard M. Holtegaard, and Carl T. Wittwer. "Mutation Scanning of the RET Protooncogene Using High-Resolution Melting Analysis." Clinical Chemistry 52, no. 1 (January 1, 2006): 138–41. http://dx.doi.org/10.1373/clinchem.2005.052951.

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Abstract Background: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. Methods: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen®, and the high-resolution melter, HR-1™, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. Results: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. Conclusion: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.

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Zhou, Luming, Lesi Wang, Robert Palais, Robert Pryor, and Carl T. Wittwer. "High-Resolution DNA Melting Analysis for Simultaneous Mutation Scanning and Genotyping in Solution." Clinical Chemistry 51, no. 10 (October 1, 2005): 1770–77. http://dx.doi.org/10.1373/clinchem.2005.054924.

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Abstract Background: High-resolution DNA melting analysis with saturation dyes for either mutation scanning of PCR products or genotyping with unlabeled probes has been reported. However, simultaneous PCR product scanning and probe genotyping in the same reaction has not been described. Methods: Asymmetric PCR was performed in the presence of unlabeled oligonucleotide probes and a saturating fluorescent DNA dye. High-resolution melting curves for samples in either capillaries (0.3 °C/s) or microtiter format (0.1 °C/s) were generated in the same containers used for amplification. Melting curves of the factor V Leiden single-nucleotide polymorphism (SNP) and several mutations in exons 10 and 11 of the cystic fibrosis transconductance regulator gene were analyzed for both PCR product and probe melting transitions. Results: Independent verification of genotype for simple SNPs was achieved by either PCR product or probe melting transitions. Two unlabeled probes in one reaction could genotype many sequence variants with simultaneous scanning of the entire PCR product. For example, analysis of both product and probe melting transitions genotyped ΔF508, ΔI507, Q493X, I506V, and F508C variants in exon 10 and G551D, G542X, and R553X variants in exon 11. Unbiased hierarchal clustering of the melting transitions identified the specific sequence variants. Conclusions: When DNA melting is performed rapidly and observed at high resolution with saturating DNA dyes, it is possible to scan for mutations and genotype at the same time within a few minutes after amplification. The method is no more complex than PCR and may reduce the need for resequencing.

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Towler, William I., Maria M. James, Stuart C. Ray, Lei Wang, Deborah Donnell, Anthony Mwatha, Laura Guay, et al. "Analysis of HIV Diversity Using a High-Resolution Melting Assay." AIDS Research and Human Retroviruses 26, no. 8 (August 2010): 913–18. http://dx.doi.org/10.1089/aid.2009.0259.

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Bignell, Patricia A., David M. Keeling, and Paul Giangrande. "Detection of F8 Mutations by High Resolution Melting (HRM) Analysis." Blood 112, no. 11 (November 16, 2008): 1226. http://dx.doi.org/10.1182/blood.v112.11.1226.1226.

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Abstract Approximately 50% of severe and all moderate or mild haemophilia A (HA) mutations are single base substitutions, small insertions or deletions with over 900 different mutations having been described. The usual way to screen these HA cases is by DNA sequencing of all 26 exons which is time consuming and expensive. We have developed the technique of HRM analysis using the Corbett Rotor Gene 6000 for successful high throughput screening of HA mutations. A target sequence was first amplified in the presence of a dsDNA intercalating fluorescent dye followed by in-tube melting analysis. Initially fluorescence is high, but diminishes as the temperature is raised and DNA dissociates into single strands. The observed ‘melting’ behaviour is characteristic of a particular DNA mutation. In our study we looked at HA mutations in 23 exons,(exons 3–26). Genomic DNA samples from 100 HA patients and carriers were amplified using primers targeting the specific exons. We detected all of the mutations by HRM analysis and each had a distinct ‘melt’ curve which enabled the carriers to be successfully screened. We detected all the HAMSTerS known mutations and our 26 novel mutations in our cohort of patients. Our results show that HRM could be used as the initial screening method followed by DNA sequencing of the amplicon where a different ‘melt’ curve was observed. It could also be used to rescreen ‘negative’ mutation patients very quickly. This technique is simpler, cheaper and quicker than DNA sequencing method. This method may therefore be particularly useful for prenatal diagnosis and carrier diagnosis.

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DANG, XIAO‐DONG, COLIN T. KELLEHER, EMMA HOWARD‐WILLIAMS, and CONOR V. MEADE. "Rapid identification of chloroplast haplotypes using High Resolution Melting analysis." Molecular Ecology Resources 12, no. 5 (July 11, 2012): 894–908. http://dx.doi.org/10.1111/j.1755-0998.2012.03164.x.

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Langaee, Taimour, Lynda Stauffer, Cheryl Galloway, Mohamed H. Solayman, and Larisa Cavallari. "Cross-Validation of High-Resolution Melting Analysis-Based Genotyping Platform." Genetic Testing and Molecular Biomarkers 21, no. 4 (April 2017): 259–64. http://dx.doi.org/10.1089/gtmb.2016.0317.

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Issa, Rahizan, Hatijah Abdul, Siti Hasmah Hashim, Valentinus H. Seradja, Nurul ‘Aishah Shaili, and Nurul Akma Mohd Hassan. "High resolution melting analysis for the differentiation of Mycobacterium species." Journal of Medical Microbiology 63, no. 10 (October 1, 2014): 1284–87. http://dx.doi.org/10.1099/jmm.0.072611-0.

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A quantitative real-time PCR (qPCR) followed by high resolution melting (HRM) analysis was developed for the differentiation of Mycobacterium species. Rapid differentiation of Mycobacterium species is necessary for the effective diagnosis and management of tuberculosis. In this study, the 16S rRNA gene was tested as the target since this has been identified as a suitable target for the identification of mycobacteria species. During the temperature gradient and primer optimization process, the melting peak (Tm) analysis was determined at a concentration of 50 ng DNA template and 0.3, 0.4 and 0.5 µM primer. The qPCR assay for the detection of other mycobacterial species was done at the Tm and primer concentration of 62 °C and 0.4 µM, respectively. The HRM analysis generated cluster patterns that were specific and sensitive to distinguished small sequence differences of the Mycobacterium species. This study suggests that the 16S rRNA-based real-time PCR followed by HRM analysis produced unique cluster patterns for species of Mycobacterium and could differentiate the closely related mycobacteria species.

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Everman, Steven, and Shiao Y. Wang. "Rapid differentiation of bacterial communities using high resolution melting analysis." Journal of Microbiological Methods 140 (September 2017): 77–81. http://dx.doi.org/10.1016/j.mimet.2017.07.006.

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Kim, Jaai, and Changsoo Lee. "Rapid fingerprinting of methanogenic communities by high-resolution melting analysis." Bioresource Technology 174 (December 2014): 321–27. http://dx.doi.org/10.1016/j.biortech.2014.10.037.

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Li, Jian-Hong, Yue-Ping Yin, He-Ping Zheng, Ming-Ying Zhong, Rui-Rui Peng, Baoxi Wang, and Xiang-Sheng Chen. "A high-resolution melting analysis for genotyping urogenital Chlamydia trachomatis." Diagnostic Microbiology and Infectious Disease 68, no. 4 (December 2010): 366–74. http://dx.doi.org/10.1016/j.diagmicrobio.2010.07.013.

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Vaughn, Cecily P., and Kojo S. J. Elenitoba-Johnson. "High-Resolution Melting Analysis for Detection of Internal Tandem Duplications." Journal of Molecular Diagnostics 6, no. 3 (August 2004): 211–16. http://dx.doi.org/10.1016/s1525-1578(10)60512-0.

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Montgomery, Jesse L., Lindsay N. Sanford, and Carl T. Wittwer. "High-resolution DNA melting analysis in clinical research and diagnostics." Expert Review of Molecular Diagnostics 10, no. 2 (March 2010): 219–40. http://dx.doi.org/10.1586/erm.09.84.

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Yimniam, Walaiporn, and Sumalee Jindadamrongwech. "Scanning for α-Hemoglobin Variants by High-Resolution Melting Analysis." Journal of Clinical Laboratory Analysis 30, no. 5 (February 18, 2016): 633–40. http://dx.doi.org/10.1002/jcla.21914.

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Shi, Minglan, Xiqing Li, Jiao Feng, Shulin Jia, Xing Xiao, Chunmei Chen, Cindy Fransisca, Liyan Xi, and Junmin Zhang. "High-resolution melting analysis assay for identification of Fonsecaea species." Journal of Clinical Laboratory Analysis 32, no. 2 (May 22, 2017): e22257. http://dx.doi.org/10.1002/jcla.22257.

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Chang, Chun-Chi, Pei-Chin Lin, Ching-Hsiung Lin, Kun-Tu Yeh, Hsiao-Yu Hung, and Jan-Gowth Chang. "Rapid identification of CYP2C8 polymorphisms by high resolution melting analysis." Clinica Chimica Acta 413, no. 1-2 (January 2012): 298–302. http://dx.doi.org/10.1016/j.cca.2011.10.005.

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Wu, Shu-Biao, Michelle G. Wirthensohn, Peter Hunt, John P. Gibson, and Margaret Sedgley. "High resolution melting analysis of almond SNPs derived from ESTs." Theoretical and Applied Genetics 118, no. 1 (September 10, 2008): 1–14. http://dx.doi.org/10.1007/s00122-008-0870-8.

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Funayama, Manabu, Hiroyuki Tomiyama, Ruey-Meei Wu, Kotaro Ogaki, Hiroyo Yoshino, Yoshikuni Mizuno, and Nobutaka Hattori. "Rapid screening of ATP13A2 variant with high-resolution melting analysis." Movement Disorders 25, no. 14 (October 25, 2010): 2434–37. http://dx.doi.org/10.1002/mds.23106.

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HARA, Masayuki, Kazuyoshi YANO, and Etsuko UTAGAWA. "Rapid High-throughput Development on High-resolution Melting (HRM) Analysis for Noroviruses." Kansenshogaku Zasshi 84, no. 3 (2010): 315–16. http://dx.doi.org/10.11150/kansenshogakuzasshi.84.315.

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Tungphatthong, Chayapol, Jutharat Somnuek, Thatree Phadungcharoen, Kornkanok Ingkaninan, Jessada Denduangboripant, and Suchada Sukrong. "DNA barcoding of species of Bacopa coupled with high-resolution melting analysis." Genome 61, no. 12 (December 2018): 867–77. http://dx.doi.org/10.1139/gen-2018-0059.

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In Thailand, there are three species of Bacopa, namely, B. monnieri, B. caroliniana, and B. floribunda. Among these species of Bacopa, B. monnieri is the only medicinal species, used for the treatment of cognitive impairment and improvement of cognitive abilities because of its bioactive constituents, bacoside A and B. However, because of the similar characteristics of these species, it is difficult to differentiate among related species, resulting in confusion during identification. For this reason, and to ensure therapeutic quality for consumers, authentication is important. In this study, the three abovementioned species of Bacopa were evaluated using barcoding coupled with high-resolution melting (Bar-HRM) analysis based on primers designed for the trnL–F sequences of the three species. The melting profiles of the trnL–F amplicons of B. caroliniana and B. floribunda were clearly different from the melting profile of the trnL–F amplicon from B. monnieri; thus, the species could be discriminated by Bar-HRM analysis. Bar-HRM was then used to authenticate commercial products in various forms. The melting curves of the six commercial samples indicated that all the tested products contained genuine B. monnieri species. This method provides an efficient and reliable authentication system for future commercial herbal products and offers a reference system for quality control.

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Zhang Tingting, 张婷婷, 侯梦迪 Hou Mengdi, 贾卓楠 Jia Zhuonan, 花双全 Hua Shuangquan, 王文杰 Wang Wenjie, and 刘绍鼎 Liu Shaoding. "基于激光的G-四链体高分辨率熔解曲线分析." Laser & Optoelectronics Progress 59, no. 7 (2022): 0717002. http://dx.doi.org/10.3788/lop202259.0717002.

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Sovová, Tereza, Barbora Křížová, Ladislav Kučera, and Jaroslava Ovesná. "Detecting soybean and milk in dairy and soy products with post-PCR high resolution melting assays." Czech Journal of Food Sciences 38, No. 4 (August 31, 2020): 209–14. http://dx.doi.org/10.17221/125/2020-cjfs.

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We have developed and validated post-PCR (polymerase chain reaction) high resolution melting (HRM) based assays that allow identifying the presence of two major allergenic foods – soybean and bovine milk – in food products. A new set of primers for PCR was developed for detection of the gene encoding the soybean protein lectin. The assay was validated using reference samples and used for the analysis of artificially prepared mixed samples of dairy and soy products of different matrices, as well as of real products available in the market (spray creams). The limits of detection (LODs) of the soybean (8 copies) and bovine milk (4 copies) assays were lower compared to LODs of other previously published PCR-based assays. The analysis of several commercial spray creams revealed an undeclared presence of soybean in one of the samples. The newly developed HRM assays are precise and robust alternatives for the control of food composition and falsification by competent authorities.

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Ng, Jacklyn W. S., Deborah C. Holt, Patiyan Andersson, and Philip M. Giffard. "DNA Concentration Can Specify DNA Melting Point in a High-Resolution Melting Analysis Master Mix." Clinical Chemistry 60, no. 2 (February 1, 2014): 414–16. http://dx.doi.org/10.1373/clinchem.2013.215582.

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Cheng, Ju-Chien, Chien-Ling Huang, Chung-Ching Lin, Chi-Ching Chen, Yi-Chih Chang, Shy-Shin Chang, and Ching-Ping Tseng. "Rapid Detection and Identification of Clinically Important Bacteria by High-Resolution Melting Analysis after Broad-Range Ribosomal RNA Real-Time PCR." Clinical Chemistry 52, no. 11 (November 1, 2006): 1997–2004. http://dx.doi.org/10.1373/clinchem.2006.069286.

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Abstract Background: Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria. Methods: We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory. Results: The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product lengths of 216 or 217 bp. Of the 25 bacterial species, we identified 11 via a 1-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a 2nd real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. In the validation assay, a 94% accuracy rate was achieved when the bacterial species were in the high-resolution melting database. Conclusions: This assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostic laboratory.

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Thomas, Holly R., Stefanie M. Percival, Bradley K. Yoder, and John M. Parant. "High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis." PLoS ONE 9, no. 12 (December 11, 2014): e114632. http://dx.doi.org/10.1371/journal.pone.0114632.

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Reed, Gudrun H., Jana O. Kent, and Carl T. Wittwer. "High-resolution DNA melting analysis for simple and efficient molecular diagnostics." Pharmacogenomics 8, no. 6 (June 2007): 597–608. http://dx.doi.org/10.2217/14622416.8.6.597.

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Zhou, L., J. Vandersteen, L. Wang, T. Fuller, M. Taylor, B. Palais, and C. T. Wittwer. "High-resolution DNA melting curve analysis to establish HLA genotypic identity." Tissue Antigens 64, no. 2 (August 2004): 156–64. http://dx.doi.org/10.1111/j.1399-0039.2004.00248.x.

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Ababneh, Mustafa, Ola Ababneh, and Mohammad Borhan Al-Zghoul. "High-resolution melting curve analysis for infectious bronchitis virus strain differentiation." Veterinary World 13, no. 3 (2020): 400–406. http://dx.doi.org/10.14202/vetworld.2020.400-406.

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Background and Aim: Belonging to the Coronaviridae family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) curve analysis. Materials and Methods: In this study, qRT-PCR was conducted on a partial fragment S1 gene followed by a high resolution melting curve analysis (qRT-PCR/HRM) on 23 IBV-positive samples in Jordan. For this assay, we utilized the most common IBV vaccine strains (Mass and 4/91) as a reference in the HRM assay. To evaluate the discrimination power of the qRT-PCR/ HRM, we did the sequencing of the partial S1 gene. Results: It was shown that HRM was able to classify IBV samples into four clusters based on the degree of similarity between their melting points: The first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the Mass-related IBV strain. Although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant II-like (IS-1494-06) IBV field strain. Finally, the fourth cluster comprised one unique sample that was found to belong to the Q1 IBV strain. Conclusion: Our developed qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains as confirmed by S1 gene nucleotide sequences, making it a rapid and cost-effective tool.

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Girault, G., P. Wattiau, M. Saqib, B. Martin, F. Vorimore, H. Singha, M. Engelsma, et al. "High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei." Infection, Genetics and Evolution 63 (September 2018): 1–4. http://dx.doi.org/10.1016/j.meegid.2018.05.004.

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