Method for Identifying HbCS Variant in Alpha Globin Genes Causing Alpha-Thalassaemia Disease

Abstract

The Haemoglobin Constant Spring (HbCS) mutation is one of the non-deletion variants with the highest frequency causing Alpha-Thalassaemia disease. High Resolution Melting analysis was developed to detect HbCS mutation on HbA2 gene effectively to decrease the alpha-thalassaemia hereditary for offsprings. Two healthy non-pregnant DNA samples and seven HbCS positive DNA samples was collected and detected by a real-time PCR SYRBGreen assay. The Realtime PCR assay was optimized based on the concentration of PCR reagents like DNA template, DMSO, Mg2+. The DNA fragment containing alleles of the mutation is amplified from the assay and will be analyzed by melting curve. According to the normalized melting curve and the temperature shifted different plot, the difference in melting curve shapes present and the genotype of each individual which is related to the disease will be indicated clearly. The HRM method was optimized and developed successfully. The condition with 5% of DMSO and 2.5 mM of MgCl2 is the best PCR condition and gave out the best separated melting curve shape in discriminating three various genotypes. This study showed that the HRM analysis is a reliable, incomplex tool and effective cost method for identifying HbCS variant in alpha globin genes and could be applied in clinical diagnostics.