2'-fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry

被引:66
作者
Ono, T [1 ]
Scalf, M [1 ]
Smith, LM [1 ]
机构
[1] UNIV WISCONSIN,DEPT CHEM,MADISON,WI 53706
基金
美国国家科学基金会;
关键词
D O I
10.1093/nar/25.22.4581
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 2' hydrogen with an electronegative group such as hydroxyl or fluorine was investigated. These 2' 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 2'-modified nucleoside triphosphates in the polymerase-directed synthesis of DNA. This would provide an avenue to the preparation of 2'-modified PCR fragments and dideoxy sequencing ladders stabilized for MALDI analysis. In this paper methods are described for the polymerase-directed synthesis of 2'-fluoro modified DNA, using commercially available 2'-fluoronucleoside triphosphates. The ability of a number of DNA and RNA polymerases to incorporate the 2'-fluoro analogs was tested. Four thermostable DNA polymerases [Pfu (exo(-)), Vent (exo(-)), Deep Vent (exo(-)) and UITma] were found that were able to incorporate 2'-fluoronucleotides with reasonable efficiency. In order to perform Sanger sequencing reactions, the enzymes' ability to incorporate dideoxy terminators in conjunction with the 2'-fluoronucleotides was evaluated. UITma DNA polymerase was found to be the best of the enzymes tested for this purpose. MALDI analysis of enzymatically produced 2'-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.
引用
收藏
页码:4581 / 4588
页数:8
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