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

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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.