Mizuno A, Katsura S
Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 Japan.
J Biol Phys. 2002 Dec;28(4):587-603. doi: 10.1023/A:1021274319649.
A conventional method of DNA sequencing can determine up to 1000 base pairs at one time. Therefore, long DNA should be cut into many short fragments that are suitable for DNA sequencing. Those fragments, however, lose their order information. If the fragments are prepared from the terminus of the long DNA, the reorganization process can be omitted. This process consists of following unit operations; manipulation of genomic DNA, fixation with a stretched form, cutting from the terminus, recovery and amplification. In these unit operations, manipulation and cutting of DNA are focused in this report. Globular transformation suppresses break down of long genome DNA and permits manipulation of large DNA. Because globular transition is reversible, the coiled DNA can be sequentially spun from the globular DNA like a spindle. Thespun DNA was successfully fixed on a glass surface in an arbitrary pattern. To prepare fragments from the stretched DNA molecule, a method to cut DNA moleculen was developed. Since most restriction enzyme requires magnesium ion for their activation, the restriction enzyme was successfully activated only when magnesium ion was electrochemically supplied.
传统的DNA测序方法一次最多能测定1000个碱基对。因此，长DNA应该被切成许多适合DNA测序的短片段。然而，这些片段会丢失它们的顺序信息。如果片段是从长DNA的末端制备的，那么重组过程可以省略。这个过程由以下单元操作组成：基因组DNA的操作、以拉伸形式固定、从末端切割、回收和扩增。在这些单元操作中，本报告重点关注DNA的操作和切割。球状转变抑制了长基因组DNA的分解，并允许对大DNA进行操作。由于球状转变是可逆的，卷曲的DNA可以像纺锤体一样从球状DNA中依次纺出。纺出的DNA成功地以任意模式固定在玻璃表面。为了从拉伸的DNA分子制备片段，开发了一种切割DNA分子的方法。由于大多数限制酶需要镁离子来激活，只有当通过电化学方式提供镁离子时，限制酶才能成功激活。
