A New Cutting Method of DNA Based on Local Activation of Restriction Enzyme

摘要

A conventional method of DNA sequencing can determine up to 1000 base pairs at one time. Long DNA should be cut into many short DNA fragments that are suitable for DNA sequencing. Those DNA fragments, however, lose their order information. If the fragments are prepared from the terminus of the long chromosomal DNA, the reorganization process can be omitted. To achieve this, a single DNA molecule should be fixed with a stretched form, and sequentially cut from the terminus to prepare fragments that keep order information. In this report, a novel stretching and cutting method for single DNA molecules are presented. The method of stretching is based on manipulation and reversible change of globular DNA molecules. The globular DNA can be easily manipulated by electrostatic force, because this transition suppresses breakdown of long DNA molecules due to shear stress accompanied with flow. A single globular DNA molecule was anchored at the tip of needle electrode of tungsten. When the globular DNA was reverted to coiled state by reducing concentrations of condensing reagents, the coiled DNA was sequentially spun from the globular DNA like a spindle. By manipulating the tip of the needle electrode, chromosomal DNA was spun and fixed on a glass surface successfully in arbitrary. To prepare fragment from the DNA molecule stretched as previously, a method to cut DNA molecules should be developed. Since most restriction enzyme requires magnesium ion for their activation, the restriction enzyme activity can be localized by controlling local concentration of magnesium ion. The local concentration of magnesium ion can be controlled electrochemically by applying a dc voltage to a needle electrode of magnesium metal. The restriction enzyme was successfully activated only when magnesium ion was electrochemically supplied.