SNP genotyping was performed using the mass array time-of-flight mass spectrometer (Sequenom Company, USA) technique. Polymerase chain reaction (PCR) and extension primers were designed by the Mass ARRAY Assay Design 3.1 software (Sequenom Company, USA). The genotyping procedures were performed using the manufacturer's iPLEX Application Guide (Sequenom Company, USA). The PCR program for amplification conditions was 94°C, 15 min; 45 cycles × (94°C, 20 s; 56°C, 30 s; 72°C, 1 min); 72°C, 5 min. All the genotyping reactions were performed in 384-well plates, and each plate included four randomly selected duplicates and 6 negative controls; double distilled water was used as the negative controls. The average concordance rate of the genotype was 99.5%.
Mass array time of flight mass spectrometer
Manufactured by Labcorp
Sourced in United States
The mass array time-of-flight mass spectrometer is an analytical instrument used to measure the mass-to-charge ratio of ions in a sample. It functions by accelerating ions through an electric field and then measuring the time it takes for the ions to reach a detector, which is proportional to their mass-to-charge ratio.
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3 protocols using mass array time of flight mass spectrometer
1
High-Throughput SNP Genotyping by Mass Spectrometry
2
DNA Extraction and Genotyping Protocol
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Human genomic DNA samples were extracted from peripheral blood (QIAamp DNA blood kit; QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. A NanoDrop 2000 spectrophotometer (Thermo Scientific, DE, USA) was used to measure the concentration. The eluted and qualified DNA samples were stored at −80°C prior to further use.
Genotyping was performed using a MassARRAY time-of-flight mass spectrometer (Sequenom, San Diego, CA, USA), as described in our previous study (18 (link)). Success rates for rs1004467, rs17115149, and rs12413409 were 98.8, 100.0, and 98.8%, respectively.
Genotyping was performed using a MassARRAY time-of-flight mass spectrometer (Sequenom, San Diego, CA, USA), as described in our previous study (18 (link)). Success rates for rs1004467, rs17115149, and rs12413409 were 98.8, 100.0, and 98.8%, respectively.
3
HLA Genotyping via Mass Spectrometry
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Seven SNPs (rs3077 at HLA-DPA1; rs9277535, rs2281388 and rs3135021 at HLA-DPB1; rs9366816 at HLA-DPB2; rs2856718 at HLA-DQB1; and rs7453920 at HLA-DQB2) were selected in total. Genotyping was performed according to the mass array time-of-flight mass spectrometer (Sequenom company, USA). Polymerase chain reaction (PCR) and extension primers were designed using MassARRAY assay design 3.1 software (Sequenom company, USA). The genotyping procedures were performed according to the manufacturer’s iPLEX application guide (Sequenom company, USA). The PCR amplification conditions included an initial precycle incubation of 94°C for 15 minutes, followed by 45 cycles of denaturation at 94°C for 20 seconds, annealing at 56°C for 30seconds, and extension at 72°C for 60seconds. All the genotyping reactions were performed in 384-well plates. Each plate included four randomly selected duplicates and six negative controls using double distilled water. The average concordance rate for the genotypes was 99.5%.
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