IPLEX® method and MassARRAY® system for genotyping the allelic variants

The extracted DNA samples were transferred to Inqaba Biotech Co, South Africa, to determine the genotypes of the allelic variants using iPLEX® and MassARRAY® platform (Sequenom MassARRAY® System, San Diego, USA). This SNP genotyping method involves an initial PCR reaction specific to the locus followed by single base extension using mass-modified primers containing terminal deoxyribonucleotides that binds immediately upstream of the polymorphism site. Using Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) Mass Spectrometry (MS), the specific mass of the amplified primer determines the SNP allele. This procedure involves the following steps: Pre-PCR for DNA (5 to 50 ng /µl) and oligonucleotides preparation, PCR for target locus amplification using designed primers, Post-PCR for purification of the reaction with SAP (Shrimp alkaline phosphatase), Single base amplification with iPLEX system, Cleaning products with resin, Deployment of amplified products on SpectroCHIPs, and Products tracking with Mass Spectrometry with appropriate MALDI-TOF matrix [35]. The specifications of the oligos for iPLEX SNP genotyping are shown in Table ​Table22.

The specifications of the oligos for iPLEX SNP genotyping