Bsp1286I (SduI) restriction enzyme

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	The PCR product (709 bp) was treated with restriction endonuclease Bsp1286I (Takara Biotechnology, Otsu, Japan), size-separated on a 2% agarose gel with ethidium bromide, and visualized with UV light. In HNA-5a-positive homozygote samples, three fragments of 297 bp, 217 bp, and 195 bp were generated; in HNA-5a-negative homozygote samples, two fragments of 412 bp and 297 bp were generated; and in HNA-5a heterozygote samples, four fragments of 412 bp, 297 bp, 217 bp, and 195 bp were generated (Fig. 2).

Protocol tips

The PCR product (709 bp) was treated with restriction endonuclease Bsp1286I (Takara Biotechnology, Otsu, Japan), size-separated on a 2% agarose gel with ethidium bromide, and visualized with UV light. In HNA-5a-positive homozygote samples, three fragments of 297 bp, 217 bp, and 195 bp were generated; in HNA-5a-negative homozygote samples, two fragments of 412 bp and 297 bp were generated; and in HNA-5a heterozygote samples, four fragments of 412 bp, 297 bp, 217 bp, and 195 bp were generated (Fig. 2).

Publication protocol

To type HNA-5a, RT and PCR-ASRA were performed according to the protocol described by Simsek et al. (18). RNA was isolated from the EDTA blood samples of neonates and heir mothers using QIAamp RNA Blood Mini kits (Qiagen GmbH, Hilden, Germany). Reverse transcription of 0.5µg of total RNA was performed in a final volume of 20µL containing 5µM random hexamer, 1 mM of each dNTP, 2 units of RNase inhibitor, and 9 units of reverse transcriptase (Bioneer, Daejeon, Korea). After incubation at 42℃ for 60 min, samples were heated for 5 min at 94℃ to terminate reactions. The primers L5 (5'-ATTTCTCTCTTTGGGAGGAGG-3') and L5A (5'-TGGGTATG TTGTGGTCGTGG-3') were used to amplify the coding region of the cDNA. The PCR product (709 bp) was treated with restriction endonuclease Bsp1286I (Takara Biotechnology, Otsu, Japan), size-separated on a 2% agarose gel with ethidium bromide, and visualized with UV light. In HNA-5a-positive homozygote samples, three fragments of 297 bp, 217 bp, and 195 bp were generated; in HNA-5a-negative homozygote samples, two fragments of 412 bp and 297 bp were generated; and in HNA-5a heterozygote samples, four fragments of 412 bp, 297 bp, 217 bp, and 195 bp were generated (Fig. 2).

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