The last decade has seen an increased demand for high-throughput DNA analysis methods. This is mainly due to the advent of the human genome sequencing project that is now nearing completion. Even though mass spectrometry did not contribute to this project, it is clear that it will have an important role in the post-genome sequencing era, in genomics and proteomics. In genomics, mainly MALDI mass spectrometry will contribute to large-scale single nucleotide polymorphism (SNP) genotyping projects. Several strategies for allele-discrimination (hybridisation, cleavage, ligation, and primer extension) have been combined with MALDI mass spectrometric detection. In practice only primer extension has been integrated for SNP genotyping at high throughput. The methods using primer extension use quite diverse strategies in order to get to grips with the intricacies of MALDI and the time-of-flight detector. Adduct formation with ubiquitous sodium and potassium is a major problem and is circumnavigated by stringent desalting or charge-neutralisation of the DNA backbone. Resolution in the mass range of 5-8 kDa is not sufficient to resolve A/T SNPs (9 Da mass difference). Thus, either a strategy where two alleles are separated by one base or more bases is implemented, or a significant portion of the primer is removed prior to mass spectrometric detection. Problems surrounding the integration of SNP genotyping by MALDI mass spectrometry at high throughput are discussed, as well as applications that go beyond standard SNP genotyping and that are more demanding, such as molecular haplotyping, DNA methylation analysis, and mutation detection. Examples are shown that illustrate the specific qualities of mass spectrometric analysis of DNA in contrast to other DNA analysis techniques.
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