Rapid characterization of DNA oligomers and genotyping of single nucleotide polymorphism using nucleotide-specific mass tags.
Using currently available MS-based methods, accurate mass measurements are essential for the characterization of DNA oligomers. However, there is a lack of specificity in mass peaks when the characterization of individual DNA species in a mass spectrum is dependent solely upon the mass-to-charge ratio (m/z). Here, we utilize nucleotide-specific tagging with stable isotopes to provide internal signatures that quantitatively display the nucleotide content of oligomer peaks in MS spectra. The characteristic mass-split patterns induced by the partially (13)C/(15)N-enriched dNTPs in DNA oligomers indicate the number of labeled precursors and in turn the base substitution in each mass peak, and provide for efficient SNP detection. Signals in mass spectra not only reflect the masses of particular DNA oligomers, but also their specific composition of particular nucleotides. The measurements of mass tags are relative in the mass-split pattern and, hence, the accuracy of the determination of nucleotide substitution is indirectly increased. For high sample throughput, (13)C/(15)N-labeled sequences of interest have been generated, excised in solution and purified for MS analysis in a single-tube format. This method can substantially improve the specificity, accuracy and efficiency of mass spectrometry in the characterization of DNA oligomers and genetic variations.
Abdi
F
F
C-ACS, BN-2, MS M888, Chemistry Division, Bioscience Division, Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87544, USA.
Bradbury
E M
EM
Doggett
N
N
Chen
X
X
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019398:Genes, BRCA1 / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D008970:Molecular Weight; D009711:Nucleotides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity; D013997:Time Factors
D009711:Nucleotides; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C024412:GGTGA-specific type II deoxyribonucleases