TY  - JOUR
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
Y1  - 2002/07/01 
JF  - Genome Research 
JO  - Genome Research 
SP  - 1135 
EP  - 1141 
DO  - 10.1101/gr.221402 
VL  - 12 
IS  - 7 
UR  - http://genome.cshlp.org/content/12/7/1135.abstract 
N2  - 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. 
ER  -