Nucleic Acids Research, Vol 25, Issue 22 4581-4588, Copyright © 1997 by Oxford University Press
T Ono, M Scalf and LM Smith
Fragmentation is a major factor limiting mass range and resolution in the
analysis of DNA by matrix-assisted laser desorption/ionization mass
spectrometry (MALDI-MS). Protonation of the nucleobase leads to base loss
and backbone cleavage by a mechanism similar to the depurination reactions
employed in the chemical degradation method of DNA sequencing. In a
previous study [Tang,W., Zhu,L. and Smith,L.M. (1997) Anal. Chem ., 69,
302-312], the stabilizing effect of substituting the 24 hydrogen with an
electronegative group such as hydroxyl or fluorine was investigated. These
24 substitutions stabilized the N-glycosidic linkage, blocking base loss
and subsequent backbone cleavage. For such chemical modifications to be of
practical significance, it would be useful to be able to employ the
corresponding 24-modified nucleoside triphosphates in the
polymerase-directed synthesis of DNA. This would provide an avenue to the
preparation of 24-modified PCR fragments and dideoxy sequencing ladders
stabilized for MALDI analysis. In this paper methods are described for the
polymerase-directed synthesis of 24- fluoro modified DNA, using
commercially available 24-fluoronucleoside triphosphates. The ability of a
number of DNA and RNA polymerases to incorporate the 24-fluoro analogs was
tested. Four thermostable DNA polymerases [Pfu (exo-), Vent (exo-), Deep
Vent (exo-) and UlTma] were found that were able to incorporate
24-fluoronucleotides with reasonable efficiency. In order to perform Sanger
sequencing reactions, the enzymes' ability to incorporate dideoxy
terminators in conjunction with the 24-fluoronucleotides was evaluated.
UlTma DNA polymerase was found to be the best of the enzymes tested for
this purpose. MALDI analysis of enzymatically produced 24-fluoro modified
DNA using the matrix 2,5-dihydroxy benzoic acid showed no base loss or
backbone fragmentation, in contrast to the extensive fragmentation evident
with unmodified DNA of the same sequence.
ARTICLES
2'-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry
Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Takahashi, N. Minakawa, and A. Matsuda Synthesis and characterization of 2'-modified-4'-thioRNA: a comprehensive comparison of nuclease stability Nucleic Acids Res., March 1, 2009; 37(4): 1353 - 1362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Thompson, M. Prescott, N. Chelebi, J. Smith, T. Brown, and G. Schmidt Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS Nucleic Acids Res., February 28, 2007; 35(4): e28 - e28. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Capodici, K. Kariko, and D. Weissman Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference J. Immunol., November 1, 2002; 169(9): 5196 - 5201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. C. Richardson, B. C. Tennant, D. J. Meyer, K. A. Richardson, P. C. Mann, G. R. Mcginty, J. L. Wolf, P. M. Zack, and R. A. Bendele An Evaluation of the Toxicities of 2' -Fluorouridine and 2'-Fluorocytidine-HCI in F344 Rats and Woodchucks (Marmota monax) Toxicol Pathol, November 1, 1999; 27(6): 607 - 617. [Abstract] [PDF]
|
|
|
|
 |
|
 J. R. Conejo, T. Parra, M. Cantero, A. Jimenez, V. Granizo, V. Gabriel de Arriba, and F. Carballo Detection of H-ras Mutations in Urine Sediments by a Mutant-enriched PCR Technique Clin. Chem., July 1, 1998; 44(7): 1570 - 1572. [Full Text] [PDF]
|
|