TY - JOUR JF - iau-tmuj JO - MEDICAL SCIENCES VL - 15 IS - 2 PY - 2005 Y1 - 2005/6/01 TI - Development of covalent reverse dot-blot technique for rapid diagnosis of two common .-thalassemia mutations: IVS-I-5 and IVS-I-110 in Iran TT - طراحی استریپ تشخیصی با بکارگیری تکنیک هیبریدسازی کووالانت معکوس DNA برای تشخیص سریع دو موتاسیون رایج IVS-I-5 و IVS-I-110 بتاتالاسمی در ایران N2 - Background: 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. Materials and methods: 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. Results: 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. Conclusion: we described a rapid and simple strategy to detect beta-thalassemia mutations based on PCR followed by reverse Dot-Blot hybridization. SP - 79 EP - 84 AU - Hashemi M, AU - Nazemi A, AU - Forouzandeh M, AD - KW - Beta-thalassemia KW - Mutation KW - Strip KW - Reverse Dot-Blot hybridization. UR - http://tmuj.iautmu.ac.ir/article-1-270-en.html ER -