Development of high performance chromatography-based gene detection system

对DNA的快速定量分析，特别是对SNP（single nucleotide polymorphism，单核苷酸多态性）检测的需求日益增加。虽然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，SNPs在色谱中的生物识别还没有得到很好的理解。在本研究中，我们提出的蛋白质静电相互作用色谱（离子交换色谱，IEC）的生物识别机制的基础上，研究了DNA的静电相互作用色谱（IEC）。对各种寡核苷酸样品进行线性梯度洗脱实验。峰值盐浓度I_R是归一化梯度斜率GH的函数。从GH I_R曲线确定结合位点B的数目。B与寡核苷酸的长度（或负电荷数）之间有很好的相关性。B值不随移动的相pH值的变化而变化，这与蛋白质的IEC不同，IEC中B值强烈依赖于移动的相pH值，而I_R值随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容易地分离，但需要另一种新的高效方法用于更大的寡核苷酸分离（SNPs检测）。

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)