Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysis
Background. According to WHO guidelines, one of the criteria for diagnosis of polycythemia vera is the presence of somatic mutations in exon 12 of the JAK2 gene, but to date there is no universally accepted simple method to analyze these mutations. We have previously proposed two methods for screeni...
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2024-01-01
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| author | T. N. Subbotina A. A. Shalyova A. I. Shevchenko E. A. Pozdysheva Ya. A. Voytsekhovskaya K. O. Mironov |
| author_facet | T. N. Subbotina A. A. Shalyova A. I. Shevchenko E. A. Pozdysheva Ya. A. Voytsekhovskaya K. O. Mironov |
| author_sort | T. N. Subbotina |
| collection | DOAJ |
| description | Background. According to WHO guidelines, one of the criteria for diagnosis of polycythemia vera is the presence of somatic mutations in exon 12 of the JAK2 gene, but to date there is no universally accepted simple method to analyze these mutations. We have previously proposed two methods for screening such mutations based on heteroduplex and HRM (High Resolution Melt) assays, which are relatively cheap and fast compared to sequencing.Aim. To analyze the sensitivity of these screening methods.Materials and methods. The study used cloned DNA samples from 6 patients with various mutations in exon 12 of the JAK2 gene that we had previously identified, as well as a clone of the corresponding wild-type DNA segment. Dilution of the cloned mutant samples with wild-type clones was performed to obtain samples with different levels of allele burden: 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 %. Heteroduplex analysis followed by PAGE (polyacrylamide gel) and HRM analysis was then performed with the diluted samples.Results. The sensitivity threshold of the heteroduplex analysis was found to be between 3.13–6.25 % allele burdens depending on the specific mutation, the sensitivity threshold of the HRM assay was 6.25–12.5 % similarly.Conclusion. Our proposed methods of heteroduplex analysis followed by PAGE and HRM-analysis for the detection of polycythemia vera-specific mutations in exon 12 of the JAK2 gene allow increasing the efficiency of using different types of sequencing and can be used as simpler and less expensive methods of preliminary screening of these mutations. |
| format | Article |
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| institution | DOAJ |
| issn | 1818-8346 2413-4023 |
| language | Russian |
| publishDate | 2024-01-01 |
| publisher | ABV-press |
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| series | Онкогематология |
| spelling | doaj-art-3fc284f3fa844d70873a22080ed63bb92025-08-20T03:01:02ZrusABV-pressОнкогематология1818-83462413-40232024-01-01191929810.17650/1818-8346-2024-19-1-92-98743Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysisT. N. Subbotina0A. A. Shalyova1A. I. Shevchenko2E. A. Pozdysheva3Ya. A. Voytsekhovskaya4K. O. Mironov5Siberian Federal University; Federal Siberian Research Clinical Center, Federal Medical and Biological AgencySiberian Federal University; Federal Siberian Research Clinical Center, Federal Medical and Biological AgencySiberian Federal University; Federal Siberian Research Clinical Center, Federal Medical and Biological AgencyCentral Research Institute of Epidemiology, Federal Service for Surveillance on Consumer Rights Protection and Human WellbeingCentral Research Institute of Epidemiology, Federal Service for Surveillance on Consumer Rights Protection and Human WellbeingCentral Research Institute of Epidemiology, Federal Service for Surveillance on Consumer Rights Protection and Human WellbeingBackground. According to WHO guidelines, one of the criteria for diagnosis of polycythemia vera is the presence of somatic mutations in exon 12 of the JAK2 gene, but to date there is no universally accepted simple method to analyze these mutations. We have previously proposed two methods for screening such mutations based on heteroduplex and HRM (High Resolution Melt) assays, which are relatively cheap and fast compared to sequencing.Aim. To analyze the sensitivity of these screening methods.Materials and methods. The study used cloned DNA samples from 6 patients with various mutations in exon 12 of the JAK2 gene that we had previously identified, as well as a clone of the corresponding wild-type DNA segment. Dilution of the cloned mutant samples with wild-type clones was performed to obtain samples with different levels of allele burden: 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 %. Heteroduplex analysis followed by PAGE (polyacrylamide gel) and HRM analysis was then performed with the diluted samples.Results. The sensitivity threshold of the heteroduplex analysis was found to be between 3.13–6.25 % allele burdens depending on the specific mutation, the sensitivity threshold of the HRM assay was 6.25–12.5 % similarly.Conclusion. Our proposed methods of heteroduplex analysis followed by PAGE and HRM-analysis for the detection of polycythemia vera-specific mutations in exon 12 of the JAK2 gene allow increasing the efficiency of using different types of sequencing and can be used as simpler and less expensive methods of preliminary screening of these mutations.https://oncohematology.abvpress.ru/ongm/article/view/901jak2 12 exonhrmheteroduplex analysis |
| spellingShingle | T. N. Subbotina A. A. Shalyova A. I. Shevchenko E. A. Pozdysheva Ya. A. Voytsekhovskaya K. O. Mironov Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysis Онкогематология jak2 12 exon hrm heteroduplex analysis |
| title | Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysis |
| title_full | Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysis |
| title_fullStr | Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysis |
| title_full_unstemmed | Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysis |
| title_short | Sensitivity evaluation of methods for screening JAK2 exon 12 mutations based on heteroduplex and HRM analysis |
| title_sort | sensitivity evaluation of methods for screening jak2 exon 12 mutations based on heteroduplex and hrm analysis |
| topic | jak2 12 exon hrm heteroduplex analysis |
| url | https://oncohematology.abvpress.ru/ongm/article/view/901 |
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