Rapid characterization of DNA oligomers and genotyping of single nucleotide polymorphism using nucleotide-specific mass tags

Abstract

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.

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

“…The failure to identify this nucleoside represents major problems to (1) precise genotyping of an SNP, (2) the identification of an insertion or a deletion, and (3) the determination of the correct genetic code of the targeted sequence. For validation of the identity of the uncalled base, a 9-bp target region containing the uncalled base was amplified using primers containing Hph I restriction sites that allow for the generation of a 10-bp fragment on digestion as previously described (Abdi et al 2001). Hph I has the recognition sequence of 5Ј.…”

“…PCR amplifications using 50% labeled nucleosides were performed as described earlier. Hph I digestion of the amplified DNA and the purification of the 10-bp fragment were achieved as described by Abdi et al 2001.…”

“…Further, we extended this approach to genotype SNPs. The incorporation of 50% 13C/ 15N-labeled nucleosides (1 : 1 molar ratio of labeled and unlabeled nucleosides) in wild-type, mutant, and heterozygous PCR products from genomic DNA provided unique mass-split patterns in MS spectra that allowed the determination of substituted patterns of SNPs (Abdi et al 2001).…”

“…In addition, the ability to measure directly the mass-to-charge (m/z) ratio of biomolecules with high accuracy made a wide range of bioanalytical applications available to MS analysis (Blackstock and Weir 1999;Pandey and Mann 2000;Yates 2000). Because of its speed, accuracy, and sensitivity, MALDI-TOF-MS has become a powerful tool for the efficient sequencing of short DNA fragments as well as genotyping of single nucleoside polymorphisms (SNPs) Smirnov 1997a, 1997b;Fei et al 1998;Laken et al 1998;Ross et al 1998;Guo 1999;Li et al 1999;Fei and Smith 2000;Griffin and Smith 2000;Sun et al 2000;Abdi et al 2001).…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…Most solid-phase-mediated methods require a clean-up because the substrates of the allele-specific reaction can produce false-positive signals and complicate genotype scoring. Examples are mass spectrometry, 12,[16][17][18][19][20][21] zip-code technology, 22,23 Illumina color beads, 24,25 Orchid SNP-IT technology 11 and all hybridization based methods. 26,27 Product detection and identification can be performed in many different ways.…”