Pcr master mix

DNA templates were subjected to either deamination or left untreated, followed by performing length extension. Briefly, a total reaction volume of 50 µl was prepared, consisting of 5 µl of A1-200 (500 nM, 200 bp), 5 µl of B1-100 (500 nM, 100 bp), 1 µl DNA template, 25 µl of 2× PCR master mix (TaKaRa Taq), and 14 µl of nuclease-free water. The mixture was subjected to an initial extension and pre-amplification step under thermocycling conditions (95 °C for 1 min, 95 °C for 15 s, 60 °C for 10 s, and 72 °C for 30 s, for 10 cycles). The resulting pre-amplified products (5 µl) were then transferred to a new tube containing a mixture of A1 primer (0.5 µM), B1 primer (0.5 µM), and PCR master mix (TaKaRa Taq pH 8.9). The mixture was subjected to a standard PCR amplification step under thermocycling conditions (95 °C for 1 min, 95 °C for 15 s, 58 °C for 10 s, and 72 °C for 30 s, for 12 cycles). The resulting PCR products were analyzed by 20% native PAGE in TBE buffer and visualized by Sybr-gold staining and Typhoon imaging system (Amersham Biosciences) at the Cy2 channel (Supplementary Fig. 2). The amplified products were then purified by 2% agarose gel using the GeneJET gel extraction kit (ThermoFisher) and submitted for Sanger sequencing by Genewiz.

Genomic DNA was extracted from 200 μl of whole blood using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. Purified DNA samples were diagnosed using nested polymerase chain reaction (PCR) and the G6PD gene was amplified using specific primers [22 (link)]. The first PCR round was performed under the following conditions: 94 °C for 1 min, followed by 38 cycles at 94 °C for 12 s, 65 °C for 30 s, and 68 °C for 6 min, and a final extension at 68 °C for 10 min. The amplification reaction was carried out in 20 μl volume reactions, including primer volumes (1 μl of forward and reverse primer, 5 pmol/μl), 10 μl of PCR master mix (Takara Bio Inc, Shiga, Japan), and 3 μl of DNA template. The amplified products from the first round were subjected to a second PCR round with primers (Fig. 5). The second-round amplification reaction was carried out in 20-μl volume reactions, including primers (1 μl of forward and reverse primer, 5 pmol/μl), 10 μl of PCR master mix (Takara Bio Inc., Shiga, Japan), and 3 μl of DNA template. The second PCR round was performed under the following conditions: 94 °C for 1 min, followed by 35 cycles at 94 °C for 12 s, 62 °C for 25 s, and 72 °C for 3 min, and a final extension at 72 °C for 5 min. Each fragment was analysed with 2 sequencing primers (Fig. 5).

Total RNA was extracted using TRIzol reagent (Invitrogen, USA). 0.5 μg RNA was reverse-transcribed with PCR diagnosis kits (TianGen, China) and random 9-mer primers (TAKARA, Japan). PCR reaction system contains 50 ng cDNA, 1 μl of each primer and 1×PCR master Mix (TAKARA). PCR cycling conditions were as follows 94 ºC for 5 min, followed by 35 cycles at 94 ºC for 30 s, 56 ºC for 30 s, and 72 ºC for 30 s with a final extension step of 10 min at 72 ºC. The expression levels of β-actin mRNA were measured as the internal control. The sequences of primers were as follows: CLDN6: forward, 5'-TTCATCGGCAACAGCATCGT-3' and reverse, 5'-GGTTATAGAAGTCCCGGATGA-3' (amplification length, 345 bp); β-actin, forward, 5'-TACCTCCCAAGTCCTGTATGAG-3' and reverse, 5'-TGAGCAGCATCAAACTGTGTAG-3' (amplification length, 180 bp). Reactions were carried out in triplicate. The results were analyzed using Quantity One 4.4.1 software (Bio-Rad Laboratories Inc, Hercules, California, USA).