Proflex 96 well pcr system

All samples were typed using the Huaxia Platinum PCR amplification kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Multiplex amplification was performed on a ProFlex 96‐well PCR System (Thermo Fisher Scientific) following the manufacturer's protocol. The reaction mix for each sample was prepared in 25 μl volume containing 10 μl of the master mix, 10 μl of primer set, 1 μl of DNA template and 4 μl of deionized water. We employed the following thermal cycler conditions: pre‐denaturation for 1 min at 95°C, followed by 26 cycles of 94°C for 3 s, 59°C for 16 s, 65°C for 29 s, then a final extension at 60°C for 5 min, and holding at 4°C. The PCR products were electrophoresed and detected on the Applied Biosystems 3500XL Genetic Analyzer (Thermo Fisher Scientific) using POP‐4 polymer. The genotype profiles were obtained by comparing with the matching allelic ladder via GeneMapper ID‐X v.1.4 (Thermo Fisher Scientific).

Genomic DNA was obtained from multiple samples, and the pediatric patient and control groups with AITD were suitable for analysis using a PCR template of 50 ng or lower. The primers for the ITM2A gene (rs1751094: A > C) are presented in Table 2. PCR amplifications were performed in 50 µL of reaction mixtures in 96‐well thin walled trays (Nippon Genetics). The reaction mixtures consisted of 10 μmol/L target‐specific primers, 150–300 ng genomic DNA, 1× buffer (60 mmol/L Tris‐Cl, 15 mmol/L ammonium sulfate, and 100 mmol/L MgCl2), 250 μmol/L dNTPs (dATP/dGTP/dCTP/dTTP, 250 μmol/L), and 1 U Taq DNA polymerase (Bioprince, Enzynomics).³³ In PCR, the extracted genomic DNA was amplified in a ProFlex 96‐Well PCR System (Thermo Fisher Scientific) using the following PCR conditions: a preliminary step of 1 cycle at 95°C for 15 min followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 90 s, and extension at 72°C for 30 s. The final extension was performed at 60°C for 30 min.²²

We genotyped 206 Guizhou Han individuals on the ProFlex 96-Well PCR System (Thermo Fisher Scientific) using the AGCU X19 kit (DXS8378, DXS7423, DXS10148, DXS10159, DXS10134, DXS7424, DXS10164, DXS10162, DXS7132, DXS10079, DXS6789, DXS101, DXS10103, DXS10101, HPRTB, DXS6809, DXS10075, DXS10074 and DXS10135) on the basis of the recommendations. We employed a total of 10 μL as the final PCR reaction volume, including 4 μL of reaction mix, 0.2μL of A-Taq DNA polymerase, 0.8 μL of template DNA, 2 μL of primers and 3 μL of sdH2O (sterile deionized H₂O). The PCR conditions for 10 cycles (95°C for 2 min, 94°C for 30 s, 60°C for 1 min and 65°C for 1 min) and 20 cycles (94°C for 30 s, 59°C for 1 min and 72°C for 1 min) and followed a final extension for 30 min at 60°C and finally holding at 4°C for preservation. Capillary electrophoresis separation of amplified products was conducted on the Applied Biosystems 3130 Genetic Analyzers (Thermo Fisher Scientific, MA, USA) with the POP7^® polymer and a 36cm capillary array. GeneMapper ID-X v.1.4 software (Thermo Fisher Scientific) was utilized to analyze the electrophoretogram and assign the genotypes of 19 X-STRs.