A research of sample preparation method for DNA sequencing utilizing a single DNA molecule micro-manipulation

传统的DNA测序方法可以分析多达约400个碱基对。因此，长DNA应该通过几种方法切割成小片段。然而，这些片段丢失了关于其顺序的信息。需要重组过程来确定整个序列。这个过程是耗时的，需要巨大的努力。如果在不损失顺序信息的情况下制备DNA片段，则可以进行更快速的测序。本研究的目的是发展一种基本的DNA分子微操作技术（拉伸固定、切割、回收），以制备不失去有序性的DNA片段。显微镜配备有YAG激光器（红外）和氮激光器（紫外）。YAG激光器用于激光镊子和聚焦区域的温度控制，氮激光器用于切割DNA。通过高灵敏度SIT（硅增强靶）相机观察单个DNA分子。荧光染料用于观察。实验上改进了染料和光学系统的条件，以获得单个DNA分子的清晰图像。主要使用λ期和T4期DNA。溶液中的布朗运动干扰了DNA的操作。采用液氮冷冻或琼脂糖凝胶凝胶法固定DNA，外加直流电场拉伸DNA分子。通过生物素-亲和素连接将DNA分子一端连接到3 μ m的珠上，由于珠固定在凝胶中，因此可以通过电场在琼脂糖凝胶中拉伸。利用氮激光聚焦在拉伸的DNA分子上，可以在任何期望的位置切割DNA链。可以将DNA片段收集到玻璃毛细管中（i.d. 15微米）。

Conventional DNA sequencing method can analyze up to about 400 base pairs. Long DNA should therefore be cut into small pieces by several methods. However, these fragments lose information on their order. Reorganization process is required to determine whole sequence. This process is time consuming and requires tremendous effort. If DNA fragments are prepared without loss of the order information, more rapid sequencing can be made. The purpose of this research is to develop a fundamental micro-manipulation technique (stretched fixation, cutting, recovery) for single DNA molecule to prepare its fragments without loss of their order.A fluorescent microscope was used to observe single DNA molecule. The microscope equipped with a YAG laser (infrared) and a nitrogen laser (ultra violet).The YAG laser was used for laser tweezers and for temperature control of focused area, and the nitrogen laser was used for cutting a DNA.Single DNA molecule was observed by a high sensitive SIT (silicon intensified target) camera. Fluorescent dyes were used for the observation. Conditions for the dye and the optical system were experimentally improved to obtain clear images of single DNA molecules. Lambda phase and T4 phase DNA were mainly used. Brownian motion in the solution interfered the DNA manipulation. Freezing the solution by liquid nitrogen or gelation by agrose gel achieved the fixation of the DNA.A dc electric field was used to stretch the DNA molecule. A DNA molecule which attached at one terminus to a 3 micrometer bead by biotin-avidin connection could be stretched in the agrose gel by the electric field since the bead was fixed in the gel. Using the nitrogen laser focusing on the stretched DNA molecule, the DNA strand could be cut at any desired positon. The DNA fragment could be collected into the glass capillary (i.d. 15 micrometer) by electrophoresis.