Optimization a high resolution melting method for detection of alpha thalassemia based on a point mutation (HbSC)

Abstract

The Haemoglobin Constant Spring (HbCS) mutation is one of the most common variants causing alpha thalassaemia disease. Normally, ARMS-PCR is used to detect the HbCS mutation which contributes in detecting the alpha thalassaemia patients and carriers, but the sensitivity and specificity is low because misdetection can occur. Therefore, the new method like the high resolution melting (HRM) is developed to overcome the limitations of ARMS-PCR. The point mutation at the termination codon of alpha 2 globin gene on human chromosome 16 was detected by a realtime PCR SYRBGreen assay. The DNA fragment containing alleles of the mutation is amplified from assay and will be analyzed by melting curve. From the difference in melting curve shapes, the genotype of each individual which is related to the disease will be indicated. The HRM method for detecting various genotypes of the mutation is optimized based on the concentration of PCR reagents such as DNA template, DMSO, Mg 2+ . According to the normalized melting curve and the temperature shifted different plot, the HRM result will be analyzed clearly. The HRM method was optimized and developed successfully. The condition with 5% of DMSO and 2.5mM of MgCl 2 is the best PCR condition. HRM analysis based on normalized and temperature shifted different plot at 78 C to 88 C will give the best separated melting curve 0 0 shape in discriminating three various genotypes. This study showed that the HRM analysis is a reliable, simple tool and effective cost method for identifying HbCS variant in alpha globin genes and could be applied in clinical diagnostics. Key words: HbCS mutation, High resolution melting, Different shaped curves