RT Journal
A1 Abdi, Fadi A.
A1 Mundt, Mark
A1 Doggett, Norman
A1 Bradbury, E. Morton
A1 Chen, Xian
T1 Validation of DNA Sequences Using Mass Spectrometry Coupled with Nucleoside Mass Tagging
JF Genome Research 
JO Genome Research 
YR 2002 
FD July 01 
VO 12 
IS 7 
SP 1135 
OP 1141 
DO 10.1101/gr.221402 
UL http://genome.cshlp.org/content/12/7/1135.abstract 
AB 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 of13C/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 selected13C/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.