Development of High Performance DNA Sequencer Using Laser-Induced Capillary Vibration Method

从人类基因组计划可以看出，分析时间的加快和灵敏度的提高将促进基础研究和应用研究。近年来，毛细管区带电泳(CZE)作为一种分离超大量组分的工具备受关注。然而，CZE没有足够灵敏的检测方法来检测超微量的分离成分，因此很难应用于DNA片段等超微量材料。我们提出了激光诱导毛细管振动法(CVL)作为CZE的高灵敏度在线检测器，并用CVL方法定量检测了DNA片段的全摩尔量。本研究旨在通过分析化学和分子生物学的研究，开发一种分离效率和灵敏度高一个数量级的DNA测序方法。今年是三年研究的最后一年。前些年，我们发展了脉冲激光诱导驻波CVL检测方法。将所建立的方法应用于DNA片段的检测，其绝对量的检测限为16amol。该方法还被应用于基因诊断，并敏感地检测到被认为与心脏病或阿尔茨海默病有关的DNA片段。这些结果提示开展基因诊断的可行性，这是预防医学和早期发现的关键。此外，我们还应用于高性能免疫诊断，探索了分离免疫结合样本和游离样本的简便方法，建立了比常规方法更灵敏、更简便的免疫分析方法。与使用荧光标记或放射性同位素的方法相比，检测阿托摩尔量的生物材料具有相同或更高的灵敏度。实现无需标记过程的超灵敏检测将为DNA分析开辟一个新的应用领域。

As can be seen from Human Genome Project, acceleration of analysis time and improvement of sensitivity will promote both fundamental and application study. In recent years, capillary zone electrophoresis (CZE) has attracted much attention as a separation tool for ultramacro amounts of components. however, CZE has no detection method sensitive enough for ultramicro amounts of separated componts, and it has been difficult to apply to ultramicro amounts of materials such as DNA fragments. We proposed capilary vibration induced by laser (CVL) method as a highly sensitive on-column detector for CZE,and attomole amounts of DNA fragments were quantitatively detected using the CVL method. In this study we aimed at developing a novel DNA sequencing method with higher separation efficiency and sensitivity by one order of magnitude by maans of analytical chemistry and molecular biology.This year is the last year of the three year research. In the previous year, we developed pulsed laser-induced stationary wave CVL detection method. We applied the developed method to detection of DNA fragments, and the detection limit of absolute amount was 16 amol. The method was also applied to gene diagnosis, and DNA fragments which was considered to have relation with heart disease or Alzheimer's disease were sensitively detected. These results suggested feasibility of development of gene diagnosis, which is a key to preventive medicine and early detection. Moreover, we applied to high performance immune diagnosis, we investigated on simple method for separating immunologically bound samples and free samples, and developed more sensitive and simpler immunoassay method than the conventional method. Attomole amounts of biological materials were detected with the same or higher sensitivity than the methods using fluorescent tags or radioisotopes. Realization of ultrasensitive detection without labeling process will lead to open a new application field in DNA analysis.