Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Apr 25;23(8):1367–1373. doi: 10.1093/nar/23.8.1367

A procedure for selective DNA alkylation and detection by mass spectrometry.

I G Gut 1, S Beck 1
PMCID: PMC306863  PMID: 7753628

Abstract

A method which improves the detectability of DNA by mass spectrometry is presented. By quantitatively alkylating the backbone of phosphorothioate oligonucleotides the problems of gas phase ion generation by matrix assisted laser desorption ionization can be controlled. We have developed a selective alkylating protocol for phosphorothioate oligonucleotides which is a facile way of generating non-ionic nucleic acids. A variety of alkylating agents was studied and their kinetics were monitored in a gel electrophoretic assay and by mass spectrometry.

Full text

PDF
1367

Images in this article

1368Image
on p.1368

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker T. R., Keough T., Dobson R. L., Riley T. A., Hasselfield J. A., Hesselberth P. E. Antisense DNA oligonucleotides. I: The use of ionspray tandem mass spectrometry for the sequence verification of methylphosphonate oligodeoxyribonucleotides. Rapid Commun Mass Spectrom. 1993 Mar;7(3):190–194. doi: 10.1002/rcm.1290070305. [DOI] [PubMed] [Google Scholar]
  2. Cosstick R., McLaughlin L. W., Eckstein F. Fluorescent labelling of tRNA and oligodeoxynucleotides using T4 RNA ligase. Nucleic Acids Res. 1984 Feb 24;12(4):1791–1810. doi: 10.1093/nar/12.4.1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Eckstein F., Gish G. Phosphorothioates in molecular biology. Trends Biochem Sci. 1989 Mar;14(3):97–100. doi: 10.1016/0968-0004(89)90130-8. [DOI] [PubMed] [Google Scholar]
  4. Gish G., Eckstein F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science. 1988 Jun 10;240(4858):1520–1522. doi: 10.1126/science.2453926. [DOI] [PubMed] [Google Scholar]
  5. Heise T. W., Yeung E. S. Spatial and temporal imaging of gas phase protein and DNA produced by matrix-assisted laser desorption. Anal Chem. 1994 Feb 1;66(3):355–361. doi: 10.1021/ac00075a008. [DOI] [PubMed] [Google Scholar]
  6. Hodges R. R., Conway N. E., McLaughlin L. W. "Post-assay" covalent labeling of phosphorothioate-containing nucleic acids with multiple fluorescent markers. Biochemistry. 1989 Jan 10;28(1):261–267. doi: 10.1021/bi00427a036. [DOI] [PubMed] [Google Scholar]
  7. Karas M., Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15;60(20):2299–2301. doi: 10.1021/ac00171a028. [DOI] [PubMed] [Google Scholar]
  8. Keough T., Baker T. R., Dobson R. L., Lacey M. P., Riley T. A., Hasselfield J. A., Hesselberth P. E. Antisense DNA oligonucleotides. II: The use of matrix-assisted laser desorption/ionization mass spectrometry for the sequence verification of methylphosphonate oligodeoxyribonucleotides. Rapid Commun Mass Spectrom. 1993 Mar;7(3):195–200. doi: 10.1002/rcm.1290070306. [DOI] [PubMed] [Google Scholar]
  9. Labeit S., Lehrach H., Goody R. S. A new method of DNA sequencing using deoxynucleoside alpha-thiotriphosphates. DNA. 1986 Apr;5(2):173–177. doi: 10.1089/dna.1986.5.173. [DOI] [PubMed] [Google Scholar]
  10. Mock K. K., Sutton C. W., Cottrell J. S. Sample immobilization protocols for matrix-assisted laser-desorption mass spectrometry. Rapid Commun Mass Spectrom. 1992 Apr;6(4):233–238. doi: 10.1002/rcm.1290060402. [DOI] [PubMed] [Google Scholar]
  11. Nakamaye K. L., Gish G., Eckstein F., Vosberg H. P. Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates. Nucleic Acids Res. 1988 Nov 11;16(21):9947–9959. doi: 10.1093/nar/16.21.9947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nordhoff E., Cramer R., Karas M., Hillenkamp F., Kirpekar F., Kristiansen K., Roepstorff P. Ion stability of nucleic acids in infrared matrix-assisted laser desorption/ionization mass spectrometry. Nucleic Acids Res. 1993 Jul 25;21(15):3347–3357. doi: 10.1093/nar/21.15.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nordhoff E., Ingendoh A., Cramer R., Overberg A., Stahl B., Karas M., Hillenkamp F., Crain P. F. Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared. Rapid Commun Mass Spectrom. 1992 Dec;6(12):771–776. doi: 10.1002/rcm.1290061212. [DOI] [PubMed] [Google Scholar]
  14. Pieles U., Zürcher W., Schär M., Moser H. E. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides. Nucleic Acids Res. 1993 Jul 11;21(14):3191–3196. doi: 10.1093/nar/21.14.3191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tang K., Allman S. L., Chen C. H., Cháng L. Y., Schell M. Matrix-assisted laser desorption/ionization of restriction enzyme-digested DNA. Rapid Commun Mass Spectrom. 1994 Feb;8(2):183–186. doi: 10.1002/rcm.1290080212. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES