Assay design 3

Genomic DNA was isolated from peripheral blood using standard protocols for genetic analysis40 (link). Variants genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by Bioyong Technologies (Beijing, China) following manufacturers’ instructions14 (link)47 (link). Locus-specific amplifying primers, and single-base extending primers, were designed using Sequenom Assay Design 3.1 software, and were synthesized and diluted as required. Primer quality was assayed using a mass spectrometric system42 (link)48 (link). Locus-specific amplification by multiplex PCR, and purification of PCR products, were conducted as previously described14 (link)47 (link)49 (link). MassARRAY Typer 4.0 software (Sequenom) was used to analyze spectrometric results and generate the genotype data of each variant50 (link). All the procedures were performed by investigators blinded to sample status, i.e., from case or control subjects. Duplicate samples, positive and negative controls, were included to confirm genotyping accuracy. Direct sequencing of the amplicons containing these variants in 8% of randomly selected samples was carried out as quality controls to test the reliability51 (link)52 (link).

Standard polymerase chain reaction (PCR) protocols were used to amplify the DNA specimens. Assay Design 3.1 software (Sequenom Inc., San Diego, CA, USA) was used to design the Primers for the PCR process. The PCR reaction was done in a total volume of 5 μL containing 1 μL DNA sample (10 ng/ μL), MgCl₂ 1.625 mM, 500 μM dNTP, PCR Buffer 1×, 1 unit HotStart Taq DNA polymerase, 0.1 μM PCR primers, and 1.8 μL ddH₂O. The PCR cycling started with an initial denaturation at 94 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 20 s, annealing at 56 °C for 30 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 3 min. SNP genotyping was determined by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) in the MassARRAY system (Sequenom, San, Diego, CA, USA). PCR reaction was performed on 384-well plates using a MassARRAY Nanodispenser (Sequenom). As quality control measures, the extracted DNA was checked for purity and concentration using ultraviolet spectrophotometer at ultraviolet (UV) readings of 260 nm and 280 nm (Beckman, USA). This was then followed by electrophoresis, PCR amplification, and cluster analysis with samples showing good scatter plots accepted for the final genotyping. Both blinded and unblinded samples were tested separately and replicated concordance rates of >99.99 %.

Blood samples were used to extract genomic DNA by a DNA isolation kit (TIANGEN, China). Next, the SNP genotypes were determined using the Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF–MS) of the MassARRAY system (Sequenom Inc., San Diego, CA, USA). The Assay Design 3.1 software (Sequenom Inc., San Diego, CA, USA) was used to design PCR primers. Genotyping was performed by the Bio Miao Biological Technology (Beijing) Co., Ltd., without any knowledge about the case or control status.