Diego Genotyping Using Restriction Enzyme and Melting Curve Analysis

Background: Diego blood group genotyping is essential in East Asian populations to prevent hemolytic reactions.Conventional PCR-restriction fragment length polymorphism requires electrophoresis.This study developed a restriction enzyme-melting curve analysis (RE-MCA) method for electrophoresis-free Diego genotyping.Methods: DNA extracted from 110 red blood cell units underwent real-time PCR.Three restriction enzymes (BsrFI, MspI, and NaeI) were added directly to PCR products for MCA.The overlapping peaks in heterozygous samples were resolved by diluting the digested products with a 0∼15% dimethyl sulfoxide (DMSO) solution for MCA.The results were validated using Sanger sequencing.Results: Only BsrFI successfully digested DI*02/DI*02 without non-specific cleavage.Although homozygotes showed distinct peaks (DI*02/DI*02: ∼80.0°C/75.8°C;DI*01/DI*01: ∼83.8°C),DI*01/DI*02 heterozygotes produced merged curves.Diluting a 10% DMSO solution successfully resolved this into three distinct peaks (∼82.0°C,∼76.8°C, and ∼71.7°C), enabling unambiguous genotype discrimination.Among the 110 samples, 100 were DI*02/DI*02 (90.9%) and 10 were DI*01/DI*02 (9.1%).RE-MCA showed 100% concordance with Sanger sequencing.Conclusion: RE-MCA is a reliable and electrophoresis-free method for Diego blood group genotyping by combining restriction enzyme digestion with melting curve analysis.(Korean J Blood Transfus 2026;37:76-88