Comparison of the sensitivity of restriction analysis and PCR with high-resolution melting curve analysis for the detection of the R882H mutation in the DNMT3A gene

DOI: https://doi.org/10.29296/24999490-2023-06-04

E.D. Kulaeva(1), E.S. Muzlaeva(1), P.V. Lipilkin(2, 3), E.V. Mashkina(1)
1-Southern Federal University, Academy of Biology and Biotechnology, Stachki Ave. 194/1, Rostov-on-Don, 344090, Russian Federation;
2-Rostov State Medical University, Department of Hematology and Transfusiology (with courses in clinical laboratory diagnostics,
genetics and laboratory genetics), per. Nakhichevansky, 29, Rostov-on-Don, 344022, Russian Federation;
3-Don State Technical University, Department of Biology and General Pathology,
pl. Gagarina, 1, Rostov-on-Don, 344000, Russian Federation

Introduction. DNMT3A is one of the most frequently mutated genes in acute myeloid leukemia (AML), and the R882H (G>A) substitution is one of the most common mutations in this gene. Restriction analysis and high resolution melting (HRM) analysis are the most common methods used to detect this mutation, but the sensitivity of these approaches varies greatly from study to study. Purpose of the study. Determine the sensitivity of commonly used methods for assessing the R882H mutation content of a sample under controlled conditions. Methods. We compared the sensitivity of restriction analysis and HRM on presynthesized DNA samples with varying levels of mutant template in the sample and provided detailed protocols for reproducing our results by other researchers. Results. We found that the detection limit of the R882H mutation in the DNMT3A gene was 20% for restriction analysis and 40% for HRM. Conclusion. The results obtained in this work are important for identifying restriction analysis and HRM as suitable methods for use in laboratory diagnosis of the R882H mutation for patients with AML with a high mutational load.
Keywords: 
DNMT3A, restriction analysis, high-resolution melting curve analysis
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