Detection of unknown deletions in alpha globin genes in alpha thalassemia carriers using Real-time PCR

Alpha-thalassemia is one of the most prevalent hemoglobin disorders in the world and it is a common hereditary condition caused by deletion of one or more α-globin genes. Common α-thalassemia deletions like 3.7 kb, 4.2 kb, 20.5 kb and Med can be detected by Multiplex PCR. There are, however, some unknown deletions that can not be detected by the mentioned method or even by direct DNA sequencing. In the present study, Real-time PCR was used to determine the presence or absence of unknown deletions. Real-time PCR was performed using intercalating dye SYBR Green I and α1, α2 and CLCN7 genes were amplified. Data analysis was conducted using comparative threshold method (ΔΔCT) for determination of Gene dosage of α1-globin and α2-globin genes. The results showed the ratio of 0.90±0.16 for normal individuals and the ratio of 0.32±0.15 for carrier samples with deletions. In addition, Melting curve analysis confirmed the specific amplification of target genes. The Real-time PCR assay is simple, rapid, and reliable. It can be applied for direct determination of unknown deletions in Alpha-thalassemia carriers.