Genescan analysis software version 3

The multiplex PCR products were purified to remove remaining primers and dNTPs through the following procedure. A 7 μL purification reaction consisted of 3 μL of PCR product, 0.8 μL of 1 U/μL of FastAP (Fermentas), and 0.2 μL of 20 u/μL ExoI (Fermentas). The reaction was incubated at 37°C for 15 min, then at 80°C for 15 min to inactivate the enzymes. The extension reaction was performed using the SNaPshot mix in an EDC-810 PCR Thermal Cycler with 1 μL of SNaPshot ready reaction mix, 0.1 μL of extension primer for each mutation and 2 μL of purified PCR products in a 6 μL total volume. Extension reaction was performed for 30 cycles of 96°C for 10 s, 52°C for 5 s, and 60°C for 30 s. Genotyping of the G6PD mutations relies on four different fluorochromes and controlled extension products sizes (final extension product sizes ranged between 22 and 38 bp). After extension, 1 μL of product was mixed with 9.5 μL of Hi-Di^™ formamide and 0.5 μL of GeneScan 120 LIZ size standard (Applied Biosystems, USA). This mixture was denatured at 95°C for 3 min and chilled immediately on ice. The fluorescently labeled products were separated by capillary electrophoresis on an ABI PRISM 3730 DNA Sequencer. Sizes of the extension products were analyzed by using the GeneScan Analysis Software version 3.7 (Applied Biosystems).