Vol. 12, Issue 7, 1135-1141, July 2002

METHODS
Validation of DNA Sequences Using Mass Spectrometry Coupled with Nucleoside Mass Tagging

¹ Analytical Chemistry Sciences (C-ACS), ² Biosciences (BN-2), MS M888, Los Alamos National Laboratory, Los Alamos New Mexico 87545, USA; ³ Department of Biological Chemistry, School of Medicine, University of California at Davis, Davis, California 95616, USA

We present a mass spectrometry (MS)-based nucleoside-specific mass-tagging method to validate genomic DNA sequences containing ambiguities not resolved by gel electrophoresis. Selected types of ¹³C/¹⁵N-labeled dNTPs are used in PCR amplification of target regions followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-MS analysis. From the mass difference between the PCR products generated with unlabeled nucleosides and products containing ¹³C/¹⁵N-labeled nucleosides, we determined the base composition of the genomic regions of interest. Two approaches were used to verify the target regions: The first approach used nucleosides partially enriched with stable isotopes to identify a single uncalled base in a gel electrophoresis-sequenced region. The second approach used mass tags with 100% heavy nucleosides to examine a GC-rich region of a polycytidine string with an unknown number of cytidines. By use of selected ¹³C/¹⁵N-labeled dNTPs (dCTPs) in PCR amplification of the target region in tandem with MALDI-TOF-MS, we determined precisely that this string contains 11 cytidines. Both approaches show the ability of our MS-based mass-tagging strategy to solve critical questions of sequence identities that might be essential in determining the proper reading frames of the targeted regions.