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Nucleic Acids Research 2006 34(3):e18; doi:10.1093/nar/gnj021
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Published online 9 February 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry

Florence Mauger, Olivier Jaunay, Valérie Chamblain, Fred Reichert1, Keith Bauer1, Ivo G. Gut* and David H. Gelfand1

Centre National de Génotypage, Bâtiment G2 2 Rue Gaston Crémieux, 91057 Evry Cedex, France 1Roche Molecular Systems Inc. 1145 Atlantic Avenue, Alameda, CA 94501, USA

*To whom correspondence should be addressed. Tel: +33 160 87 84 00; Fax: +33 160 87 83 83; Email: ivo.gut{at}cng.fr

Received November 21, 2005. Revised January 19, 2006. Accepted January 19, 2006.

Single nucleotide polymorphisms (SNPs) are now widely used for many DNA analysis applications such as linkage disequilibrium mapping, pharmacogenomics and traceability. Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3' terminus. The method was tested on four SNPs each with a different combination of nucleotides. The advantage over other mass spectrometry-based SNP genotyping assays is that this one only requires a PCR, a primer extension reaction with a universal extension mix and an inexpensive facile cleavage reaction, which makes it overall very cost effective and easy in handling.


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