SNP genotyping
Teaching SNPs how to fly: Typing SNPs using MALDI-TOF mass spectrometry
It has already been over ten years since the first successful analysis of DNA using MALDI-TOF (matrix-assisted laser desorption ionisation time of flight) mass spectrometry. In the meantime MALDI-TOF MS has become one of several available methods for the analysis of single nucleotide polymorphisms (SNPs), in addition to assays based on microarrays and beads. In a recent review in PLoS Genetics, Jiannis Ragoussis and colleagues of the Wellcome Trust Centre for Human Genetics in Oxford outline the uses of MALDI-TOF MS in genomics research, and include an insightful description the applicability of this method to SNP analysis.
Several MALDI-TOF MS-based assays are available for SNP genotyping, including methods based on an initial PCR amplification coupled with a primer extension or an invader reaction, and those based on a hybridization procedure followed by an exonuclease reaction. In each of these cases the reaction products are detected using MALDI-TOF MS, using methods such as the GOOD or PinPoint assays, the GenoSNIP from Bruker Daltonics, or the MassEXTEND from Sequenom.
The MassEXTEND assay was the first homogeneous reaction carried out in single wells using unmodified primers, making it an ideal solution for higher-throughput genotyping requirements. An important current limitation of this method is the inherent need to multiplex the reactions in order to achieve an acceptably high throughput. The primary bottleneck in this regard is the PCR step, as multiplex PCR reactions create several challenges in terms of maintaining amplification specificity for competing primer sets, as well as minimizing interactions between these different primers.
Additional SNP analysis technologies with comparable throughput include ABI's SNPlex and Beckman Coulter's SNPStream systems, which can both multiplex up to 48 reactions in 384-well format. Although it remains to be seen whether MALDI-TOF MS will become a suitable technology for the genome-wide analysis of SNPs, it is clearly appropriate for the high-throughput analysis of smaller numbers of SNPs, and is already being used for this purpose in clinical diagnosics applications.
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