High-performance liquid chromatographic analysis of DNA composition and DNA modification by chloroacetaldehyde.
High-performance liquid chromatographic analysis of DNA composition and DNA modification by chloroacetaldehyde.
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DNA 组成和氯乙醛 DNA 修饰的高效液相色谱分析。
DOI：
10.1016/s0021-9673(00)90558-2
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Landes,JP
中科院分区：
文献类型：
--
作者：
Singhal,RP;Landes,JP
在不同的色谱条件下，基于硅胶的十八烷基（C18）柱上，涉及与基质的疏水相互作用的DNA水解产物的常见和修饰的脱氧核糖核苷的分离进行了研究。描述了一种用于分析DNA组成的新方法。它涉及RNA污染物的去除和DNA的酶促水解，首先为脱氧核糖核苷单磷酸，然后将后者去磷酸化为脱氧核糖核苷。寻求水解条件以避免dA和dC残留物分别脱氨基为dI和dU污染物。洗脱这些污染物和文物（核糖核苷衍生自RNA）的描述与脱氧核糖核苷的洗脱。在选定的分离条件下，在一个高效液相色谱柱上，在室温下18分钟内实现了15μg DNA样品的水解产物的色谱分离。使用二极管阵列检测器可以增强对微量改性成分（和污染物）的检测。这种技术的力量在于它能够准确地表征和定量DNA结构中存在的修饰物质的量（小于所有其他残基的2%）。描述了来源于原核生物（大肠杆菌B）和真核生物（鲑鱼精子）的DNA的组成分析的实例。通过市售软件提供不同核苷的定量（校准图）详情，用于峰面积积分，并描述洗脱缓冲液中核苷的光谱特性，用于通过其他方法进行定量。这里示出的组合物分析的应用，通过化学手段探测溶液中的DNA构象，同时使用氯乙醛作为修饰剂。
The separation of common and modified deoxyribonucleosides derived from DNA hydrolyzates was examined under different chromatographic conditions on silica-based octadecyl (C18) columns, involving hydrophobic interactions with the matrix. A novel method for the analysis of the DNA composition is described. It involves the removal of RNA contaminants and enzymatic hydrolysis of DNA, first to deoxyribonucleoside monophosphates and then dephosphorylation of the latter to deoxyribonucleosides. Hydrolysis conditions were sought to avoid deamination of dA and dC residues to dI and dU contaminants, respectively. Elution of these contaminants and the artifacts (ribonucleosides derived from RNA) is described in relation to the elution of deoxyribonucleosides. Chromatographic separation of the hydrolyzate derived from a 15μg sample of DNA under selected separation conditions and on one high-performance liquid chromatographic column is achieved in 18 min at room temperature. Detection of modified components (and contaminants) present in minute amounts is enhanced with the use of a diode-array detector. The power of this technique lies in its ability to characterize and quantitate accurately the amount of modified species present in the DNA structure (less than 2% of all the other residues). Examples of the composition analysis of DNA derived from a prokaryote (Escherichia coliB) and a eukaryote (salmon sperm) are described. Details of quantitation (calibration graphs) of different nucleosides are furnished for peak-area integration by commercially available software, and spectral properties of the nucleoside in the elution buffer are described for quantitation by other means. Application of the composition analysis is shown here for probing the DNA conformation in solution by chemical means, while using chloroacetaldehyde as the modifying agent.