Detection of the JAK2V617F Mutation in Myeloproliferative Disorders by Melting Curve Analysis Using the LightCycler System
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Abstract Context.—A specific mutation, JAK2V617F, was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory. Objective.—We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing. Design.—Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind). Results.—The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2V617F amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2V617F was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia. Conclusions.—These findings demonstrate the suitability of this assay for identifying JAK2V617F in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2V617F in archived specimens provides a new tool for studying the prognostic significance of this mutation.Keywords:
Melting curve analysis
High Resolution Melt
Amplicon
Myeloproliferative Disorders
Myeloproliferative neoplasm
Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.
High Resolution Melt
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Sanger sequencing
Multiplex
Melting curve analysis
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Abstract Homozygosity for the α-thalassaemia Southeast Asian (α-SEA) and Filipino β 0 -thalassaemia (β-FIL) deletions can cause serious complications leading to foetal death or life-long blood transfusions. A rapid and accurate molecular detection assay is essential in populations where the deletions are common. In this study, gap-polymerase chain reaction (PCR) with high resolution melting (HRM) analysis was developed to detect both the large deletions. Melting curves at 86.9 ± 0.1 °C were generated by normal individuals without the α-SEA deletion, 84.7 ± 0.1 °C by homozygous α-SEA deletion individuals and two melting curves at 84.7 ± 0.1 °C and 86.9 ± 0.1 °C by α-SEA deletion carriers. Normal individuals without the β-FIL deletion produce amplicons with a melting temperature (Tm) at 74.6 ± 0.1 °C, homozygous β-FIL individuals produce amplicons with Tm at 73.6 ± 0.1 °C and heterozygous β-FIL individuals generate two amplicons with Tm at 73.6 ± 0.1 °C and 74.6 ± 0.1 °C. Evaluation using blinded tests on 220 DNA samples showed 100% sensitivity and specificity. The developed assays are sensitive and specific for rapid molecular and prenatal diagnosis for the α-SEA and β-FIL deletions.
High Resolution Melt
Beta thalassemia
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Abstract Context.—A specific mutation, JAK2V617F, was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory. Objective.—We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing. Design.—Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind). Results.—The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2V617F amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2V617F was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia. Conclusions.—These findings demonstrate the suitability of this assay for identifying JAK2V617F in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2V617F in archived specimens provides a new tool for studying the prognostic significance of this mutation.
Melting curve analysis
High Resolution Melt
Amplicon
Myeloproliferative Disorders
Myeloproliferative neoplasm
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Abstract Background: X-linked Charcot-Marie-Tooth type 1 disease has been associated with 280 mutations in the GJB1 [gap junction protein, beta 1, 32kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked)] gene. High-resolution melting analysis with an automated instrument can be used to scan DNA for alterations, but its use in X-linked disorders has not been described. Methods: A 96-well LightScanner for high resolution melting analysis was used to scan amplicons of the GJB1 gene. All mutations reported in this study had been confirmed previously by sequence analysis. DNA samples were amplified with the double-stranded DNA-binding dye LC Green Plus. Melting curves were analyzed as fluorescence difference plots. The shift and curve shapes of melting profiles were used to distinguish controls from patient samples. Results: The method detected each of the 23 mutations used in this study. Eighteen known mutations provided validation of the high-resolution melting method and a further 5 mutations were identified in a blind study. Altered fluorescence difference curves for all the mutations were easily distinguished from the wild-type melting profile. Conclusion: High-resolution melting analysis is a simple, sensitive, and cost-efficient alternative method to scan for gene mutations in the GJB1 gene. The technology has the potential to reduce sequencing burden and would be suitable for mutation screening of exons of large multiexon genes that have been discovered to be associated with Charcot Marie Tooth neuropathy.
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Myeloproliferative neoplasms (MPNs) are a group of clonal disorders characterized by hyperproliferation of hematologic cell lines and have been associated with tyrosine kinase JAK2-V617F mutations. Secondary acute myeloid leukemia (sAML) is a known complication of JAK2-V617F+ MPNs and bears a poor prognosis. Although the evolution of a JAK2-V617F+ MPN to a mixed-lineage leukemia has been reported in the pediatric population, no evolutions into sAML have been described. We present a case of a one-year-old girl diagnosed with JAK2-V617F+ MPN with evolution into sAML. Despite initial morphologic remission, she eventually relapsed and succumbed to her disease.
Myeloproliferative neoplasm
Myeloproliferative Disorders
Lineage (genetic)
Girl
JAK2 V617F
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A real-time polymerase chain reaction (PCR) assay utilizing high resolution melt (HRM) curve analysis was developed and tested for the monitoring of Vibrio cholerae in water samples. The assay utilized previously published primers that are specific to regions of the V. cholerae ompWand ctxAB genes, allowing it to differentiate between toxigenic and non-toxigenic strains. TheompW and ctxAB primers amplify target regions of 588 and 564 bp in length (respectively) and the amplicons could be accurately identified using HRM curve analysis. High resolution melt curve analysis provided additional accuracy for the determination of amplicon melting temperatures, and allowed amplification of the two targets in a multiplex reaction. Two laboratories employed the assay to analyse 178 water samples obtained from diverse environmental water sources, for the presence of V. cholerae. The assay was found to be a rapid, highly accurate, sensitive and cost effective method for the detection and distinction between toxigenic and non-toxigenic V. cholerae strains in water.
Key words: Vibrio cholerae, high resolution melt, real-time polymerase chain reaction (PCR).
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Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis.
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Primer (cosmetics)
Melting curve analysis
COLD-PCR
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Multiplex
Melting curve analysis
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Enumeration
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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.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.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.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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Abstract Context.—Real-time polymerase chain reaction technologies have replaced many of the more labor-intense methods in the molecular diagnostics laboratory. Similarly, melt curve analysis can provide a rapid means of mutation screening. Objective.—To determine if real-time polymerase chain reaction and melt curve analysis using the SmartCycler II could be used as a screening tool for 3 common mutations in BRCA1 and BRCA2. Design.—Real-time polymerase chain reaction amplification with SYBR Green I detection was performed on DNA from cell lines known to carry the 185delAG or 5382insC mutation in BRCA1 or the 6174delT mutation in BRCA2. The melting temperatures and the melt curves were analyzed for differences between wild-type DNA and cell lines that were heterozygous for each mutation. Results.—Significant differences were present in the melt curves for each of the mutations compared with those of the wild-type sequences. The melt curve for the 185delAG mutation showed a separate peak at a lower temperature, which represented the melting temperature of the heteroduplex. For the 6174delT mutation, the melt curve had a shoulder at a lower temperature, while the melt curve for the 5382insC mutation was shifted to the left and was broader than that for the wild-type sequences. Conclusions.—High-resolution melt curve analysis is a quick, reliable method for identifying mutations due to small deletions or insertions. As a proof of principle, we used this assay to identify the 3 most common BRCA1 and BRCA2 mutations in the Ashkenazi Jewish population.
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High Resolution Melt
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