Sauer Sascha, Reinhardt Richard, Lehrach Hans, Gut Ivo G
Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Ihnestrasse 63-73, 14195 Berlin-Dahlem, Germany.
Nat Protoc. 2006;1(4):1761-71. doi: 10.1038/nprot.2006.257.
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.
基质辅助激光解吸电离（MALDI）质谱已发展成为一种分析核酸的强大方法。在此，我们提供基于聚合酶链反应（PCR）和引物延伸以产生等位基因特异性产物，通过MALDI进行单核苷酸多态性（SNP）基因分型的方案。此外，我们展示了在MALDI分析之前用于引物延伸产物样品制备的三种不同方法，并讨论了它们潜在的应用领域。第一种方法，即“GOOD”分析，是一种无需纯化的程序，它使用DNA修饰化学方法，包括对延伸引物中硫代磷酸酯键进行烷基化。另外两种方法使用固相萃取或微阵列纯化来纯化引物延伸产物。根据各种方法的反应步骤，这些方案大约需要6 - 8小时。
