Development Of Dna Controls For Detection Of Beta-Thalassemia Mutations Commonly Found In Asian

Introduction: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of beta-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe beta-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for beta-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly.Methods: Four recombinant beta-globin plasmids, including a normal sequence and three different mutant panels covering 10 common beta-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis.Results: We demonstrated the applicability of established positive DNA controls for beta-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant beta-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant beta-globin plasmids, are required for detecting 10 common beta-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis.Conclusion: The beta-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of beta-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.