Nucleic Acids Research, 2002, Vol. 30, No. 14 e67
© 2002 Oxford University Press
Re-sequencing of multiple single nucleotide polymorphisms by liquid chromatography–electrospray ionization mass spectrometry
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a, A-6020 Innsbruck, Austria, 1 Stanford Genome Technology Center, 855 California Avenue, Palo Alto, CA 94304, USA and 2 Instrumental Analysis and Bioanalysis, Saarland University, Im Stadtwald, 66123 Saarbrücken, Germany
*To whom correspondence should be addressed. Tel: +49 681 302 2433; Fax: +49 681 302 2963; Email: christian.huber{at}mx.uni-saarland.de
Allelic discrimination of single nucleotide polymorphisms (SNPs) and, particularly, determination of the phase of multiple variations are of utmost importance in genetics. The physicochemical separation of alleles by completely denaturing ion-pair reversed-phase high-performance liquid chromatography and their on-line sequence determination by electrospray ionization mass spectrometry is demonstrated. Simultaneous genotyping of two and three simple sequence polymorphisms contained within 73–114 bp was accomplished with low femtomolar amounts of unpurified amplicons from polymerase chain reaction. Determination of allelic composition is enabled by the high accuracy (better than 0.019%) of intact mass measurements or by comparative sequencing using gas-phase fragmentation and tandem mass spectrometry in combination with fully automated, computer-aided data interpretation.