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

单DNA分子显微操作DNA测序样品制备方法研究

基本信息

批准号：
04454036
负责人：
MIZUNO Akira
金额：
$ 3.97万
依托单位：
Toyohashi University of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

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.

常规的DNA测序方法可以分析多达400个碱基对。因此，应通过几种方法将长DNA切成小块。但是，这些碎片丢失了有关其命令的信息。需要重组过程以确定整个序列。这个过程很耗时，需要巨大的努力。如果准备DNA片段而不会丢失订单信息，则可以进行更快的测序。这项研究的目的是为单个DNA分子开发一种基本的微型操作技术（拉伸固定，切割，恢复），以准备其片段而不会损失其秩序。荧光显微镜用于观察单个DNA分子。配备了YAG激光器（红外线）和氮激光（Ultra Violet）的显微镜。YAG激光器用于激光镊子和聚焦区域的温度控制，并将氮激光器用于切割DNA分子的DNA Molecule。荧光染料用于观察。实验改善了染料和光学系统的条件，以获得单个DNA分子的清晰图像。 Lambda相和T4相DNA主要使用。溶液中的布朗运动干扰了DNA操作。通过液氮或凝胶化将溶液冻结，使DNA的固定固定。ADC电场用于拉伸DNA分子。一个DNA分子在一个末端连接到生物素-Avidin连接到3微米珠的DNA分子可以通过电场在Agrose凝胶中拉伸，因为珠子固定在凝胶中。使用以拉伸DNA分子为重点的氮激光器，可以在任何所需的正面下切开DNA链。可以通过电泳将DNA片段收集到玻璃毛细管（I.D. 15千分尺）中。