Abstract
Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purification-free procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6–8 hours.
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Acknowledgements
We thank K.-D. Klöppel, K. Neff, P. Kepper, A. Smyra, J. Müller, K. Guse, C. Weidner, A. Dahl and B. M. Kliem for discussions and help. Supported by the European Union (LSHG-CT-2004-503155, MolTools), the German National Genome Research Network (01GR0414), GABI-Génoplante 2 (0313098C) and the Max-Planck Society.
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Sauer, S., Reinhardt, R., Lehrach, H. et al. Single-nucleotide polymorphisms: analysis by mass spectrometry. Nat Protoc 1, 1761–1771 (2006). https://doi.org/10.1038/nprot.2006.257
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DOI: https://doi.org/10.1038/nprot.2006.257
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