Development of covalent reverse dot-blot technique for rapid diagnosis of two common beta-thalassemia mutations: IVS-I-5 and IVS-I-110 in Iran

ABSTRACT

Beta-thalassemia is an autosomal recessive disorder that most commonly caused by point mutations in the beta-globin gene. IVS-I-5 and IVS-I-110 are the most frequent mutations found among Iranians comprising 12% of all mutations. In this study, we develop the implementation of the reverse Dot- Blot [RDB] hybridization technique as a rapid and simple method for the detection of the two common mutations in the beta-globin gene. Total genomic DNA was extracted and PCR with specific primer [forward and reverse primer] was performed on region of beta-globin gene consisting the two mutations. Labeled dNTPs with DIG-II-dUTP were used in PCR mixture therefore PCR product contained DIG labeling formed. The oligonucleotide probe was immobilized onto a Biodyne C nylon membrane via activation membrane. The strips were hybridized with 10 microliters of denatured PCR product labeled to DIG at 42°C for 60 minutes. After blocking strip with the blocking buffer, the strip exposed to 5 unit anti-DIG antibody conjugates with alkaline phosphatase for 30 minutes at room temperature. The color detection was performed with nitroblue tetrazolium salt [NBT/BCIP] substrate for 120 minutes. The presence of a particular DNA sequence was detected by the appearance of a dot on the membrane. Normal individuals [N/N] showed dots with each wild-type sequence but not with any mutant probe. Heterozygotic individuals showed the appearance of a single mutation dot in addition to all the normal dots and homozygous individuals revealed dots with each mutant probe but not with any wild-type sequence. We described a rapid and simple strategy to detect beta-thalassemia mutations based on PCR followed by reverse Dot-Blot hybridization