Development of high performance chromatography-based gene detection system

基于高效色谱的基因检测系统的开发

• 批准号: 14550739
• 负责人: YAMAMOTO Shuichi
• 金额: $ 2.18万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14550739/
• 关键词: DNA; chromatography; biorecognition; melt curve; conformational changes; SNPs; electrostatic interaction; size exclusion curve

There is an increasing need for rapid and quantitative analysis for DNA, especially for SNP (single nucleotide polymorphism) detection. Although DNA chips or microarrays are considered to be very promising detection methods, chromatography is also suited for this purpose as fully automated HPLC systems are already available. However, biorecognition in chromatography of SNPs is not well understood compared with that of other biomolecules. In this study the biorecognition mechanism in electrostatic interaction chromatography (ion-exchange chromatography, IEC) of DNA was investigated on the basis of our model proposed for IEC of proteins. Linear gradient elution experiments were carried out for various oligonucleotides as samples. The peak salt concentration I_R was measured as a function of normalized gradient slop GH. From the GH I_R curves, the number of binding sites B was determined. There was a good correlation between B and the length of oligonucleotides (or the number of negative charges). Th B values did not change with the mobile phase pH. This is different from IEC of proteins where B values strongly depend on the mobile phase pH. The I_R value increased with increasing molecular weight of oligonucleotides. Although small oligonucleotides having a single nucleotide difference can be easily resolved by conventional gradient elution IEC, another new efficient method is needed for larger oligonucleotide separation (SNPs detection)

对 DNA 快速定量分析的需求日益增长，尤其是 SNP（单核苷酸多态性）检测。尽管 DNA 芯片或微阵列被认为是非常有前途的检测方法，但色谱法也适用于此目的，因为全自动 HPLC 系统已经可用。然而，与其他生物分子相比，SNP 色谱法的生物识别尚不清楚。在本研究中，基于我们提出的蛋白质 IEC 模型，研究了 DNA 静电相互作用色谱（离子交换色谱，IEC）的生物识别机制。以各种寡核苷酸为样品进行线性梯度洗脱实验。峰值盐浓度 I_R 被测量为归一化梯度斜率 GH 的函数。从GH I_R曲线，确定结合位点B的数量。 B与寡核苷酸的长度（或负电荷的数量）之间存在良好的相关性。 Th B 值不随流动相 pH 值变化。这与蛋白质的 IEC 不同，其中 B 值很大程度上取决于流动相 pH 值。 I_R值随着寡核苷酸分子量的增加而增加。虽然具有单核苷酸差异的小寡核苷酸可以通过常规梯度洗脱 IEC 轻松解析，但需要另一种新的有效方法来分离更大的寡核苷酸（SNP 检测）