Thongperm MunkongdeeTiwaporn NualkaewNattrika BuasuwanNurmeeha HinnaKittiphong PaiboonsukwongOrapan SripichaiSaovaros SvastiPranee WinichagoonSuthat FucharoenNatee JearawiriyapaisarnMahidol UniversityFaculty of Medicine, Siriraj Hospital, Mahidol UniversityNational Institutes of Health (NIH)2020-08-252020-08-252020-01-01International Journal of Laboratory Hematology. (2020)1751553X175155212-s2.0-85088398905https://repository.li.mahidol.ac.th/handle/123456789/57759© 2020 John Wiley & Sons Ltd Introduction: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of β-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe β-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 β-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. Methods: Four recombinant β-globin plasmids, including a normal sequence and three different mutant panels covering 10 common β-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 β-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant β-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 β-globin plasmids, are required for detecting 10 common β-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. Conclusion: The β-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 β-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1111/ijlh.13292