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.

Figure 1

A schematic representation of our strategy for efficient and high throughput SNP detection. The synthetic PCR primers are biotinylated and the restriction recognition site of HphI is included at the 5′-end of each primer. HphI has the recognition sequence of 5′…GGTGA (N₈)…3′, which dictates the restriction cleavage at the 8th nucleotide from the 5′ end or 7th from the 3′ end. The nucleotides, N, representing the complimentary sequences of the genomic DNA are included in the primer sequences. After PCR amplification and labeling with ¹³C/¹⁵N-labeled nucleotides, x, streptavidin magneSphere paramagnetic beads are added to the solution to capture the biotinylated PCR products through biotin–streptavidin interactions. The PCR products linked to the beads are washed and then subjected to the HphI restriction digestion, and the target sequence containing SNP site(s), HphI mid-digest, or a, is released in solution. The biotin-labeled end of the digest portions [HphI recognition sequence plus (N)₈/(N)₇ flanking regions], b and c, are attached to the paramagnetic beads and trapped with a magnesphere technology magnetic separation stand. The labeled HphI mid-digest, a, is then desalted via a nitrocellulose membrane and dried for MALDI-TOF analysis. In MALDI-TOF spectra, the ¹³C/¹⁵N-labeled nucleotides, x, will induce a mass shift in product a, away from the molecular mass of the unlabeled product. This mass shift gives the number of labeled nucleotides incorporated in the amplified product.

Figure 2

Negative-ion MALDI-TOF mass spectra of HphI mid-digests of PCR products amplified from the mutant-type of a site at exon 1186 of BRCA-1 labeled with (A) 50% ¹³C/¹⁵N-dGTP and (B) 50% ¹³C/¹⁵N-dCTP. + Na⁺ indicates the sodium adducts. 12Da or 15Da indicates a mass tag for a ¹³C/¹⁵N-labeled dCTP or dGTP. + Na⁺ or 23Da indicates sodium adducts.

Figure 3

Negative-ion MALDI-TOF mass spectra of HphI mid-digests of PCR products labeled with 50% ¹³C/¹⁵N-dGTP amplified from (A) the wild-type, (B) the mutant-type of a site at exon 1186 of BRCA-1. + Na⁺ indicates the sodium adducts. 12 Da or 15Da indicates a mass tag for a ¹³C/¹⁵N-labeled dCTP or dGTP.

Figure 4

Negative-ion MALDI-TOF mass spectra of HphI mid-digests of PCR products labeled with 50% ¹³C/¹⁵N-dATP amplified from (A) the wild-type, (B) the mutant-type of a site at exon 1186 of BRCA-1. + Na⁺ or 23Da indicate sodium adducts. +K⁺ or 39 Da indicate potassium adducts. 15Da indicates the magnitude of the mass tag for ¹³C/¹⁵N-labeled dATP.

Figure 5

Negative-ion MALDI-TOF MS spectra of the unlabeled and individually 99% ¹³C/¹⁵N-dNTP-labeled HphI mid-digest amplified from the wild-type gene with (A) unlabeled dNTPs, (B) 99% ¹³C/¹⁵N-labeled dATP, (C) 99% ¹³C/¹⁵N-labeled dTTP, (D) 99% ¹³C/¹⁵N-labeled dCTP, (E) 99% ¹³C/¹⁵N-labeled dGTP. Asterisks indicate the mass peaks of the (–) strains derived from the experimentally measured masses of the (+) strains due to the weakness of the (–) strains. + Na⁺ or 23 Da indicate sodium adducts. +K⁺ or 39 Da indicate potassium adducts. All spectra were obtained using an accelerating voltage of 20 kV, a grid voltage of 95%, with 128 average scans, and an extraction delay time of 600 ns.

Figure 6

Negative-ion MALDI-TOF MS spectra of the unlabeled and individually 99% ¹³C/¹⁵N-dNTP-labeled HphI mid-digest PCR amplified from the mutant-form gene with (A) unlabeled dNTPs, (B) 99% ¹³C/¹⁵N-labeled dATP, (C) 99% ¹³C/¹⁵N-labeled dTTP, (D) 99% ¹³C/¹⁵N-labeled dCTP, (E) 99% ¹³C/¹⁵N-labeled dGTP. + Na⁺ or 23 Da indicate sodium adducts. + K⁺ or 39 Da indicate potassium adducts. All spectra were obtained using an accelerating voltage of 20 kV, a grid voltage of 95%, with 128 average scans and an extraction delay time of 600 ns.

Figure 7

Negative-ion MALDI-TOF mass spectra of HphI mid-digests of PCR products labeled with (A) 0%, (B) 50% and (C) 100% ¹³C/¹⁵N-dGTP amplified from the genomic DNA containing a heterozygous site at exon 1186 of BRCA-1. WU, MU, WL and ML designate the unlabeled wild-type, the unlabeled mutant-form, the labeled wild-type and the labeled mutant-form, respectively. +Na⁺ or 23 Da indicate sodium adducts. +K⁺ or 39 Da indicate potassium adducts. 15Da indicates the magnitude of the mass tag for ¹³C/¹⁵N-labeled dGTP.

Figure 8

Negative-ion MALDI-TOF mass spectra of the 31 bp PCR products obtained from amplification with (A) the unlabeled dNTP precursors, (B) 100% ¹³C/¹⁵N-dATP, (C) both 100% ¹³C/¹⁵N-dATP and ¹³C/¹⁵N-dTTP. +K⁺ or 39Da indicate potassium adducts. UL or L designate to a unlabeled or labeled strain, respectively.