Validation of DNA sequences using mass spectrometry coupled with nucleoside mass tagging.
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 (13)C/(15)N-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 (13)C/(15)N-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 (13)C/(15)N-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.
Abdi
Fadi A
FA
Analytical Chemistry Sciences, MS M888, Los Alamos National Laboratory, Los Alamos New Mexico 87545, USA.
Mundt
Mark
M
Doggett
Norman
N
Bradbury
E Morton
EM
Chen
Xian
X
D001483:Base Sequence; D002247:Carbon Isotopes / Q000032:analysis; D002885:Chromosomes, Human, Pair 16 / Q000235:genetics; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics; D003845:Deoxycytosine Nucleotides / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000032:analysis; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002247:Carbon Isotopes; D003845:Deoxycytosine Nucleotides; D009587:Nitrogen Isotopes