DEVELOPMENT OF COVALENT REVERSE DOT-BLOT TECHNIQUE FOR RAPID DIAGNOSIS OF TWO COMMON Β-THALASSEMIA MUTATIONS: IVS-I-5 AND IVS-I-110 IN IRAN

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HASHEMI M.; NAZEMI A.; FOROUZANDEH M.

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Journal: Medical Sciences Journal of Islamic Azad University,Tehran Medical Branch Year:2005 | Volume:15 | Issue:2 Page(s): 79-84

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Title

DEVELOPMENT OF COVALENT REVERSE DOT-BLOT TECHNIQUE FOR RAPID DIAGNOSIS OF TWO COMMON Β-THALASSEMIA MUTATIONS: IVS-I-5 AND IVS-I-110 IN IRAN

Author(s)

HASHEMI M. | NAZEMI A. | FOROUZANDEH M. | Issue Writer Certificate

Keywords

BETA-THALASSEMIAQ4

MUTATIONQ3

STRIPQ3

REVERSE DOT-BLOT HYBRIDIZATIONQ4

Abstract

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.

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APA: Copy

HASHEMI, M., NAZEMI, A., & FOROUZANDEH, M.. (2005). DEVELOPMENT OF COVALENT REVERSE DOT-BLOT TECHNIQUE FOR RAPID DIAGNOSIS OF TWO COMMON ?-THALASSEMIA MUTATIONS: IVS-I-5 AND IVS-I-110 IN IRAN. MEDICAL SCIENCES JOURNAL OF ISLAMIC AZAD UNIVERSITY, 15(2), 79-84. SID. https://sid.ir/paper/98664/en

Vancouver: Copy

HASHEMI M., NAZEMI A., FOROUZANDEH M.. DEVELOPMENT OF COVALENT REVERSE DOT-BLOT TECHNIQUE FOR RAPID DIAGNOSIS OF TWO COMMON ?-THALASSEMIA MUTATIONS: IVS-I-5 AND IVS-I-110 IN IRAN. MEDICAL SCIENCES JOURNAL OF ISLAMIC AZAD UNIVERSITY[Internet]. 2005;15(2):79-84. Available from: https://sid.ir/paper/98664/en

IEEE: Copy

M. HASHEMI, A. NAZEMI, and M. FOROUZANDEH, “DEVELOPMENT OF COVALENT REVERSE DOT-BLOT TECHNIQUE FOR RAPID DIAGNOSIS OF TWO COMMON ?-THALASSEMIA MUTATIONS: IVS-I-5 AND IVS-I-110 IN IRAN,” MEDICAL SCIENCES JOURNAL OF ISLAMIC AZAD UNIVERSITY, vol. 15, no. 2, pp. 79–84, 2005, [Online]. Available: https://sid.ir/paper/98664/en

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