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

Abstract

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.The major challenge in biology is to understand the function of living cells at the molecular level. To address this challenge, a major effort is now underway to sequence the genomes of a range of eukaryotic and prokaryotic organisms. Through these efforts, the draft sequence of the human genome has been completed very recently (Genome Consortium 2001). This draft sequence provides the foundation for the identification of gene sequences and the ultimate understanding of the functions of their protein products in both human diversity and disease development (Godovac-Zimmermann and Brown 2001). To accomplish these objectives, an accurate genome sequence is essential for the subsequent studies of gene functions; however, the current draft of the human genome sequence contains many short regions that are still unresolved mainly because of difficulties in sequencing these regions or artifacts caused by gel electrophoresis. These regions are found throughout the genome and are either primarily GC rich or consist of long repeats (Genome Consortium 2001). They form "hot spots" in the genome that are difficult to sequence and often result in inconsistent data on resequencing. It is essential that these inconsistencies be resolved accurately to prevent problems in interpreting the expression of genes in these regions.In the past decade, mass spectrometry (MS) has become an essential tool in both DNA and protein analyses, as well as the key technology in the emerging fields of proteomics and functional genomics (Godovac-Zimmermann and Brown 2001). Developed in the late 1980s, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-MS (Karas and Hillenkamp 1988) provided fast and accurate measureme...

Genotyping single nucleotide polymorphisms by mass spectrometry

“…Very recently, this method was applied to sequence a short GC-rich fragment, and for the identification of a previously unidentified base. This experiment demonstrated its applicability for sequence validation and for short-gap closures in the human genome sequence draft (Abdi et al, 2002). However, it is still limited to small fragments, like all other techniques for the analysis of whole PCR products, and the presence or absence of an SNP is determined, but not its position.…”

Examination of Hypochlorous Acid-Induced Damage to Cytosine Residues in a CpG Dinucleotide in DNA

“…Central to understanding the impact of HOCl-mediated damage on epigenetic patterns is information on the reactivity of cytosine in a CpG dinucleotide in duplex DNA. In order to probe the reactivity of a specific cytosine residue, we have adopted an approach referred to by our group and others as mass tagging – . In this approach, individual residues can be distinguished by increasing their mass with stable isotopes.…”

Use of 13C as an Indirect Tag in 15N Specifically Labeled Nucleosides. Syntheses of [8-13C-1,7,NH2-15N3]Adenosine, -Guanosine, and Their Deoxy Analogues

“…A significant advantage of this use of 15 N as a probe is that it does not perturb the structure while giving straightforward spectra . In a related but different method, uniformly 15 N-labeled nucleic acid samples have proven to be highly effective for structure determination by 1 H NMR, although nonhelical regions and larger molecules can leave assignment ambiguities for which specific labeling by chemical synthesis provides a clear resolution. − Further, multilabeled nucleosides and nucleic acids also provide useful mass tags for mass spectroscopic work. − …”

“…[13][14][15] Further, multilabeled nucleosides and nucleic acids also provide useful mass tags for mass spectroscopic work. [16][17][18][19][20] To facilitate 15 N NMR studies of specifically labeled DNA and RNA, we have developed methods for efficient synthesis of a variety of 15 N-multilabeled purine nucleosides. [21][22][23][24] To permit spectral differentiation of two multilabeled nucleotides, we introduced the use of a 13 C tag at the purine C2 position to distinguish the neighboring 15 N1, 15 N3, and the guanine 15 NH 2 from those in an untagged nucleotide based on the substantial 13 C- 15 N couplings (6-23 Hz).…”