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Nucleic Acids Research, 2002, Vol. 30, No. 5 e22
© 2002 Oxford University Press

Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry

Sascha Sauer1,2, David H. Gelfand3, Francis Boussicault2, Keith Bauer3, Fred Reichert3 and Ivo G. Gut2,*

1Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie, Takustrasse 3, 14195 Berlin-Dahlem, Germany, 2Centre National de Génotypage, Bâtiment G2, 2 Rue Gaston Crémieux, 91057 Evry Cedex, France and 3Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501, USA

In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or {alpha}-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and {alpha}-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.

* To whom correspondence should be addressed. Tel: +33 160 87 83 59; Fax: +33 160 87 83 83; Email: ivogut{at}cng.fr


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