Читать книгу Genotyping by Sequencing for Crop Improvement - Группа авторов - Страница 54

MALDI MS (matrix‐assisted laser desorption/ionization mass spectrometry) allows for the quick study of high molecular weight biomolecules (e.g. DNA, proteins, peptides, polysaccharides, and other organic compounds) and other chemical substances (e.g. polymers, macromolecules, and hybrid materials). It produces single‐charged ions and has a high detection accuracy and sensitivity. Sequenom incorporates matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry technique for highly accurate, low‐throughput SNP genotyping. MALDI‐TOF‐based SNP genotyping platforms take the advantage of differences in mass between allele‐specific primer and extension products. During the MALDI process, analyte molecules are mixed with low molecular weight organic molecules like α‐cyano‐4‐hydroxycinnamic acid (CHCA) and 2,5‐dihydroxybenzoic acid (DHB) called matrix and the mixture is crystallized when dried over SpectroCHIP. Then the crystallized matrix‐analyte mixture (a large quantity of matrix is used) is irradiated with a laser beam with an emitting wavelength range in UV or mid IR range. The irradiation causes desorption and ionization of analyte molecules in the form of singly protonated ions. The system uses a TOF mass analyzer. The protonated ions are then accelerated with uniform translational energy on application of the electric field. Then the charged ions are moved to the drift region which is free of electric field. The velocity of charged ions in the drift region is determined by the mass to charge ratio of the ions which in turn is calculated by measuring time required by the ions to travel through the flight tube (Figure 2.4).

Figure 2.4 Schematic illustration of work‐flow in a MALDI‐TOF MS

Source: The figure is reproduced from Singhal et al., 2015, figure 01(p.03), available with Creative Commons 4.0

For SNP genotyping, the target regions of the sample (5–10 ng) are amplified for each assay within the multiplex. The PCR product is mixed with the shrimp alkaline phosphatase (SAP) which dephosphorylates any residual dNTPs, which might interfere with the allele‐specific termination at the later stage. The amplification process is followed by a post‐PCR primer extension where an allele‐specific primer is annealed at the adjacent position of the SNP site. The extension reaction cocktail contains four terminating nucleotides (ddNTPs). The extension involves the addition of a single nucleotide and hence alleles are differentiated by the molecular mass of the extension product. The extension products are then conditioned with the ion‐exchange resin and a nano dispensing device loads a few nanoliters of sample product to the prefabricated matrix loaded chip array (384‐pad spectroCHIP) (Oath et al. 2009). The SpectroCHIP is then loaded to the MALDI‐TOF MS analyzer for the analysis.