Sequencing Oligonucleotides by Exonuclease Digestion and Delayed Extraction Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Smirnov, Igor P.; Roskey, Mark; Juhász, Péter; Takach, Edward J.; Martin, Stephen A.; Haff, Lawrence A.

doi:10.1006/abio.1996.0243

Cited by 74 publications

(61 citation statements)

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“…Oligonucleotide sequence can be determined by partial enzymatic digestion and the position of some modified bases can be revealed Previously, the sequence of oligonucleotides could be revealed by controlled digestion with exonucleases (26,29,33,34,36). It is generally presumed that such exonucleases work only on single-stranded oligonucleotides.…”

Section: Loss Of Purines From Dna Can Reduce Substantially the Thermomentioning

confidence: 99%

“…Less work has been done with MALDI-TOF-MS to examine oligonucleotides, and in particular oligonucleotides with non-standard bases (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Indeed, Guengerich and coworkers established previously capillary electrophoresis and MALDI-TOF-MS as the standard for rigorous characterization of synthetic oligonucleotides containing carcinogen adducts (35).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cui

Theruvathu

Farrel

et al. 2008

Analytical Biochemistry

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Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of structural damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this paper, we describe the application of MALDI-TOF-MS to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with non-standard synthesis, deprotection or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and utilization.

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Section: Loss Of Purines From Dna Can Reduce Substantially the Thermomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cui

Theruvathu

Farrel

et al. 2008

Analytical Biochemistry

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“…The [b 3 -P] ϩ , [b 5 -P] ϩ and [b 7 -P] ϩ ions were observed at m/z 860.1, 1477.3 and 2094.6, respectively. Other members of the [b n -P] ϩ ions were however found to be redundant in masses with y n ϩ ions, such as [b 2 -P] ϩ /y 2 ; [b 4 -P] ϩ /y 4 ; [b 6 -P] ϩ /y 6 ions. Other series of fragment ions, such as w n ϩ and y n ϩ , did not produce similar ions.…”

Section: Fast Dissociationmentioning

confidence: 99%

“…They can serve as useful tools in molecular biology, as gene probes for diagnosis, or as potential drugs interacting at the level of nucleic acids. Typical short-chain DNA sequencing methods involve the use of the enzymatic or chemical cleavage algorithms [1][2][3] to produce DNA ladder products. The DNA fragments generated are then separated and identified by HPLC or gel electrophoresis.…”

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confidence: 99%

A study of fast and metastable dissociations of adenine-thymine binary-base oligonucleotides by using positive-ion MALDI-TOF mass spectrometry

Chan

Fung

2002

J. Am. Soc. Mass Spectrom.

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In the present study, fast and metastable dissociations of a number of adenine-thymine binary-base oligonucleotides under the conditions of UV matrix-assisted laser desorption/ ionization mass spectrometry were investigated. 2-Aminobenzoic acid/ammonium fluoride (ABA/NH 4 F) matrix system was used. The spectra obtained under metastable and fast dissociation conditions exhibit distinctive dissociation products. From the post-source-decay analysis, all oligonucleotides underwent predominantly metastable dissociations at the 3' C-O linkages to form [a n -B] ϩ and w n ϩ complimentary ion series. Based on the present results, the so-called "[w n ϩ80] ϩ " ions were postulated to be the complimentary [z (8Ϫn) AH] ϩ ions rather than the expected phosphate rearrangement products. In addition, these oligonucleotides were found to generate fast dissociation products of b n ϩ , d n ϩ , w n S tructural characterization of short-chain nucleic acids has recently attracted much attention. They can serve as useful tools in molecular biology, as gene probes for diagnosis, or as potential drugs interacting at the level of nucleic acids. Typical short-chain DNA sequencing methods involve the use of the enzymatic or chemical cleavage algorithms [1][2][3] to produce DNA ladder products. The DNA fragments generated are then separated and identified by HPLC or gel electrophoresis. Structural verification of oligonucleotides using these methods is time-consuming and is sometimes impossible for modified oligonucleotides. Recent developments in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry [4 -9] have stimulated interest in replacing HPLC/gel electrophoresis by mass spectrometry in order to increase the speed and accuracy of the sequence determination. However, this approach still needs the time-consuming chemical or enzymatic reactions for producing sequence specific fragment ladder. A potentially more efficient method would be to obtain the DNA sequence information from fragmentation patterns in mass spectrometry through dissociation. In the present study, we attempt to evaluate the potential use of two rather direct mass spectrometric sequencing methods, namely fast and metastable dissociations for DNA sequence analysis.Fast fragmentation differs from metastable dissociation at the time of bond cleavage after the excitation of the precursor ions. In mass spectrometry, fragments are produced in a much shorter time as compared to the ion extraction time of the mass spectrometer. In the case of time-of-flight mass spectrometer, these fragment ions are therefore registered at their true masses in both linear and reflectron mode of detection. Metastable fragments, on the other hand, are usually formed at a much longer time scale (i.e., Ͼ500 ns) and are formed beyond the ion source region and before reaching the detector. Since the metastable ions are formed with the same velocity of the precursor ions, they are registered at the same mass-to-charge ratio of the precursor ion in a linear TOF instrum...

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“…The other is to do sequencing with DNA ladders. Pieles et al 24 and Smirnov et al 25 used time-dependent sequential excision of oligonucleotides by exonuclease and MALDI to obtain sequencing information on short oligonucleotides. McLafferty and his colleagues 26 obtained sequencing information by using collisioninduced dissociation and Fourier transform mass spectrometry for short oligonucleotides.…”

mentioning

confidence: 99%

Matrix-assisted Laser Desorption/Ionization for Sequencing Single-stranded and Double-stranded DNA

Taranenko¹,

Chung²,

Zhu³

et al. 1997

Rapid Commun. Mass Spectrom.

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The DNA sequence of a single-stranded and double-stranded template was determined. The templates were sequenced using the chain termination method and cycle sequencing method and detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sequencing products were analyzed successfully without the laborious and expensive methods for removal of the template. Direct sequencing of the double-stranded template was achieved with minimal post-reaction purifications, which could be extremely important for mutation analysis and clinical diagnosis. A systematic study of the mechanisms and kinetics of sequencing reactions was also performed. The details of this analysis and directions for future improvements of the quality of sequencing are presented.

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Sequencing Oligonucleotides by Exonuclease Digestion and Delayed Extraction Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Cited by 74 publications

References 0 publications

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A study of fast and metastable dissociations of adenine-thymine binary-base oligonucleotides by using positive-ion MALDI-TOF mass spectrometry

Matrix-assisted Laser Desorption/Ionization for Sequencing Single-stranded and Double-stranded DNA

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