Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry

Li, Jia; Butler, John M.; Tan, Yan Fei; Lin, Hua; Royer, Stephanie; Ohler, Lynne; Shaler, Thomas A.; Hunter, Joanna M.; Pollart, Daniel J.; Monforte, Joseph; Becker, Christopher H.

doi:10.1002/(sici)1522-2683(19990101)20:6<1258::aid-elps1258>3.0.co;2-v

Cited by 89 publications

(71 citation statements)

References 26 publications

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“…Stable isotope-enriched nucleotide precursors, >99% 13 C and 15 N ( 13 C/ 15 N)-labeled dNTP, were purchased from Martek Biosciences Corporation (Columbia, MD). We have separated four types of 13 C/ 15 N-labeled nucleotides by reverse-phase HPLC (Vydac C18 reverse phase 218TP54 HPLC column).…”

Section: Chemicals and Enzymesmentioning

confidence: 99%

“…Whereas several MALDI-TOF MS-based methods have been introduced for genotyping SNPs, the primary strategy has been to generate SNP-containing products through primer extension for MS analysis (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Haff and Smirnov invented a PinPoint assay based on the annealing of a primer to the 5′-end upstream of the target polymorphic site (14,15).…”

Section: Introductionmentioning

confidence: 99%

“…Here we report an extension of this strategy for the characterization of DNA oligomers and the detection of SNPs in a more efficient and accurate manner. In our new approach, partial 13 C/ 15 N-labeled dNTPs carry unique mass tags that were used to trace a particular type of nucleotide in DNA fragments or ladders. The number of labeled nucleotides in the target sequence is determined by the mass shift between the 13 C/ 15 N-labeled and unlabeled HphI digests.…”

Section: Introductionmentioning

confidence: 99%

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

Abdi¹

2001

Nucleic Acids Research

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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.

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Section: Chemicals and Enzymesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Abdi¹

2001

Nucleic Acids Research

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“…Hence, a considerable number of different methodologies for screening for polymorphisms and for determining individual allelic states have been developed, which have been reviewed recently [6,7]. Matrix-assisted laser desorption/ionization mass spectrometry MALDI-MS [8,9] and electrospray ionization mass spectrometry (ESI-MS) [10 -12] have been successfully applied to the investigation of sequence variations in nucleic acids.…”

mentioning

confidence: 99%

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

Oberacher¹,

Parson²,

Oefner³

et al. 2004

J. Am. Soc. Mass Spectrom.

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An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably resequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product. (J Am Soc Mass Spectrom 2004, 15, 510 -522)

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“…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).…”

mentioning

confidence: 99%

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

et al. 2002

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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...

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Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry

Cited by 89 publications

References 26 publications

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

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

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

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

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