Genotyping single-nucleotide polymorphisms by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry
ABSTRACT In recent years matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) has emerged as a very powerful method for genotyping single nucleotide polymorphisms. The accuracy, speed of data accumulation, and data structure are the major features of MALDI. Several SNP genotyping methods have been implemented with a high degree of automation and are being applied for large-scale association studies. Most methods for SNP genotyping using MALDI mass spectrometric detection and their potential application for high-throughput are reviewed here.
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- "The protocol includes an initial PCR amplification of the region surrounding the sequence variation of interest, followed by the addition of a primer with mass-modified terminators that anneals immediately upstream of the polymorphic site and produces a specific single-base extension of the product complementary to the SNV (Gabriel et al. 2009). The mass difference of the single-base extension products enables allelic discrimination, which is performed by MALDI-TOF mass spectrometry (Sauer and Gut, 2002). Several SNVs of interest can be amplified simultaneously, a process known as 'multiplexing'. "
ABSTRACT: SUMMARY The epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of common gp60 subtypes of four Cryptosporidium species that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) and gp60 subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four different Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. felis) were detected in humans; the most common ones were C. hominis subtype Ib, and C. parvum IIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.Parasitology 11/2013; 141(4):1-10. DOI:10.1017/S0031182013001807 · 2.35 Impact Factor
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ABSTRACT: We have directly measured thermal lensing in a continuous-wave Nd-doped GdVO4 self-Raman laser under diode pumping at 808 and 879 nm. Thermal lensing increased significantly when generating first-Stokes emission, this most likely originating from impurity absorption and/or excited-state absorption at high pump powers. Pumping at 879 nm significantly reduced thermal lens effects in the laser crystal compared with pumping at 808 nm.Applied Physics B 07/2012; 108(1). DOI:10.1007/s00340-012-4919-7 · 1.63 Impact Factor