Comparative review of diagnostic techniques for DNA methylation analysis.

INTRODUCTION

DNA methylation is a crucial epigenetic modification regulating gene expression, essential for embryonic development, cellular differentiation, and disease susceptibility. Aberrant methylation is associated with cancer, neurodegenerative and cardiovascular diseases, and imprinting disorders, highlighting the need for reliable biomarkers.

AREAS COVERED: This review summarizes six widely used DNA methylation analysis techniques: Methylation-Specific PCR (MSP), quantitative (qMSP), Methylation-Specific MLPA (MS- MLPA), Methylation-Sensitive High-Resolution Melting (MS-HRM), pyrosequencing, MassARRAY, and digital droplet PCR (ddPCR). Their principles, clinical applications, strengths, and limitations are discussed, focusing on sensitivity, specificity, cost, and laboratory requirements. MS-HRM is cost-effective and highly sensitive for clinical screening; MS- MLPA enables multiplexed detection of methylation and copy number changes; pyrosequencing and MassARRAY offer quantitative CpG-level resolution; and ddPCR allows absolute quantification in low-DNA or mosaic samples.

EXPERT OPINION: These techniques offer important advances for epigenetic diagnostics, yet further validation, protocol standardization, and simplification are needed for routine clinical use. Choosing the optimal method depends on study goals, sample type, and clinical needs. Integrating these approaches into standardized workflows can enhance early disease detection, therapeutic monitoring, and personalized epigenetic interventions, supporting precision medicine.