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Introduction: In Malaysia, β-thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR-incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β-thalassaemia mutation detection. ARMS allows single-mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β-thalassaemia mutations can be identified in samples with low DNA concentrations., Methods: Genotype identification of common β-thalassaemia mutations in Malays was carried out using Taqman genotyping assays., Results: Results show that the Taqman assays allow mutation detection with DNA template concentrations as low as 2-100 ng. In addition, consistent reproducibility was observed in the Taqman assays when repeated eight times and at different time intervals., Conclusion: The developed sensitive Taqman assays allow molecular characterization of β-thalassaemia mutations in samples with low DNA concentrations. The Taqman genotyping assays have potential as a diagnostic tool for foetal blood, chorionic villi or pre-implantation genetic diagnosis where DNA is limited and precious., (© 2014 John Wiley & Sons Ltd.)

Introduction: Dried blood spots (DBS) are currently the recommended sample collection method for newborn screening programmes in America. Early diagnosis of beta-thalassaemia screening is essential as it provides an added advantage especially in sickle cell disease. Beta-thalassaemia frequency is high in many poor countries, and the cost of using commercial DNA extraction kits can be prohibitive. Our study assessed three methods that use minimal reagents and materials to extract DNA from DBS for beta-thalassaemia identification., Methods: The methods assessed in this study were Tris-EDTA (TE) buffer-based method by Bereczky et al. (American Journal of Tropical Medicine and Hygiene 72, 2005, 249), NaCL/NaOH/Sodium dodecyl sulphate (SDS) method by Huang et al. (Human Genetics 84, 1990, 129) and NaOH method by Zhou et al. (Analytical Biochemistry 354, 2006, 159). Extracted DNA was amplified for three common beta-thalassaemia mutations in Malaysia., Results: Amplicons derived from TE buffer-based method were very faint and almost nonexistent while the NaCl/NaOH/SDS method did not produce any visible amplicons. The extraction using NaOH method produced visible bands that were comparable to the standard method using extraction kit., Conclusion: The NaOH method is a simple method that uses minimal equipment and reagents that make it labour- and cost-effective. This method could be adopted by poorer countries to extract DNA for beta-thalassaemia mutation characterization., (© 2011 Blackwell Publishing Ltd.)