Diagnosis of beta-globin gene mutations in single blastomere by reverse dot blot hybridization

Abstract

BACKGROUND: Reverse dot blot hybridization (RDB) has been employed for prenatal diagnosis (PND) of beta thalassemia due to the variety of point mutation of beta globin gene. This study try to apply this technique performed preimplantation genetic diagnosis (PGD) for beta thalassemia. The quality and quantity of PCR product is the key factor for successful hybridization of RDB. PCR reactions were modified for single blastomere to provide adequate PCR product for hybridization. METHODS: Two-step (nested) PCR was performed to amplify beta globin gene. The genomic DNA was serial diluted for determining the minimum DNA concentration could be amplified. One hundred and six single blastomeres were subjected to genotype. The optimization of annealing temperature, changing nested primer and comparing lysis method was evaluated by genotyping of 25, 50 and 256 single blastomeres respectively. Amplified product was diagnosed beta globin mutation for RDB. RESULTS:The minimum DNA concentration successful amplified was 12.5 pg. Twenty-eight of 106 (26.4%) single blastomeres were successful amplified. The optimized annealing temperature was 58 ํC. Amplification efficiency of changing location of nested PCR primer was 22.0%. The optimized primer concentration was 0.1 micrometre. Comparing five lysis methods (i) boiling in water at 94 ํC for 15 (ii) 30 min, (III) incubation in an alkali lysis buffer for 30 min at 94 ํC or (iv) at 65 C for 10 min, and (v) boiling in TE buffer at 95 ํC for 10 min. the amplification percentages were formed to be 58, 10.8, 0, 0 and 55.4% respectively. Applications of reverse dot blot hybridization to detect beta globin gene mutation of amplified DNA from 10 pg DNA of which known mutation, it was found that the detection of beta globin gene mutations were all correct. Amplified DNA from single blastomere successful hybridized. CONCLUSION : At the present study, amplification efficiency of beta globin gene in single blastomere is quite low, so that it is not appropriate for clinical applications.