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

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Abstract

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.

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About the authors

Elizaveta D. Kulaeva

Southern Federal University, Academy of Biology and Biotechnology

Email: ekulaeva@sfedu.ru
ORCID iD: 0000-0001-5886-7975

Research Assistant, Research Laboratory «Developmental Biology and Genome Organization» of Genetics Department

Russian Federation, Stachki Ave. 194/1, Rostov-on-Don, 344090

Elizaveta S. Muzlaeva

Southern Federal University, Academy of Biology and Biotechnology

Author for correspondence.
Email: ekulaeva@sfedu.ru
ORCID iD: 0000-0002-4344-4221

Bachelor student, Human and Animal Genetics Research Laboratory

Russian Federation, Stachki Ave. 194/1, Rostov-on-Don, 344090

Pavel V. Lipilkin

Rostov State Medical University, Department of Hematology and Transfusiology (with courses in clinical laboratory diagnostics, genetics and laboratory genetics); Don State Technical University, Department of Biology and General Pathology

Email: plipilkin@donstu.ru
ORCID iD: 0000-0002-3220-2753

Graduate student, Department of Hematology and Transfusiology (with courses of clinical laboratory diagnostics, genetics and laboratory genetics), Senior Lecturer, Department of Biology and General Pathology

Russian Federation, per. Nakhichevansky, 29, Rostov-on-Don, 344022; pl. Gagarina, 1, Rostov-on-Don, 344000

Elena V. Mashkina

Southern Federal University, Academy of Biology and Biotechnology

Email: lenmash@sfedu.ru
ORCID iD: 0000-0002-4424-9508

Head of Research Laboratory «Human and Animal Genetics», Doctor of Biological Sciences, Professor

Russian Federation, Stachki Ave. 194/1, Rostov-on-Don, 344090

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Supplementary files

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2. Fig. 1. Results of restriction analysis to identify the R882H mutation. wt-DNA wild type, mut - mutant DNA, 5–50 - samples with 5–50% mutant DNA in wild type DNA

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3. Fig. 2. Melting curve plot for wild type (0%) and R882H (100%) homozygotes and R882H heterozygotes (50%). The X axis is the melting temperature, °C. Y axis - reporter dye fluorescence signal, relative fluorescence units

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4. Fig. 3. Comparison of melting temperatures of samples with different DNA contents with the R882H mutation. The X axis is the content of mutant DNA in the sample, %; along the Y axis - melting temperature, °C

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