Ono T, Scalf M, Smith L M
Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Nucleic Acids Res. 1997 Nov 15;25(22):4581-8. doi: 10.1093/nar/25.22.4581.
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.
碎片化是基质辅助激光解吸/电离质谱法（MALDI-MS）分析DNA时限制质量范围和分辨率的主要因素。核碱基的质子化会导致碱基丢失和主链断裂，其机制类似于DNA测序化学降解方法中使用的脱嘌呤反应。在先前的一项研究中[Tang，W.，Zhu，L.和Smith，L.M.（1997年）《分析化学》，69，302 - 312]，研究了用羟基或氟等电负性基团取代2'-氢的稳定化作用。这些2'-取代稳定了N-糖苷键，阻止了碱基丢失和随后的主链断裂。为了使这种化学修饰具有实际意义，能够在DNA的聚合酶导向合成中使用相应的2'-修饰核苷三磷酸将是有用的。这将为制备用于MALDI分析的稳定化2'-修饰PCR片段和双脱氧测序梯提供一条途径。本文描述了使用市售的2'-氟核苷三磷酸进行聚合酶导向合成2'-氟修饰DNA的方法。测试了多种DNA和RNA聚合酶掺入2'-氟类似物的能力。发现四种热稳定DNA聚合酶[Pfu（exo-）、Vent（exo-）、Deep Vent（exo-）和UlTma]能够以合理的效率掺入2'-氟核苷酸。为了进行桑格测序反应，评估了这些酶与2'-氟核苷酸一起掺入双脱氧终止剂的能力。发现UlTma DNA聚合酶是为此目的测试的最佳酶。与相同序列的未修饰DNA明显的广泛碎片化相比，使用基质2,5-二羟基苯甲酸对酶促产生的2'-氟修饰DNA进行MALDI分析未显示碱基丢失或主链碎片化。
