Fig. 1

Steps in genotyping using MassARRAY® iPLEX System. A schematic of the genotype reaction of an A-to-G SNP. a. Locus-specific amplification reaction b. Treatment with SAP enzyme to neutralize unincorporated dNTPs. c. Locus-specific primer extension reaction (iPLEX assay). In this reaction, an oligonucleotide primer anneals immediately upstream of the polymorphic site being genotyped, the primer and amplified target DNA are incubated with mass-modified dideoxynucleotide terminators. The primer extension is made according to the sequence of the variant site. d. The products of the reactions are spotted on a spectroCHIP. The CHIP is placed into the mass spectrometer and each spot is then shot with a laser under vacuum by the matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) method. A laser beam serves as desorption and ionization source in MALDI mass spectrometry. Once the sample molecules are vaporized and ionized, they are transferred electrostatically into a time-of-flight mass spectrometer (TOF-MS), where they individually detected. The mass of the extended primer is determined. e. Sequenom supplies software that automatically translates the mass of the observed primers into a genotype for each reaction