Dissection, acquisition and amplification of targeted position of electrostatically stretched DNA

The authors have developed a DNA sequencing method based on molecular surgery, where DNA strands are stretched under a high-intensity electrostatic field, positioned onto a solid surface by dielectrophoresis, and then a targeted position is mechanically dissected under a microscope, picked up and recovered. Because thousands of molecules are stretched and aligned with one end straight on the electrode edge, as many fragments from the same location can be dissected simultaneously. The method enables the sequencing of known position on the DNA, so tedious re-construction processes in the present shot-gun sequencing can be eliminated. It is found that the mechanically dissected fragment can be recovered without loss. However, molecular damage due to mechanical dissection prohibits the ligation of adapter oligonucleotide, which is necessary for the amplification of the acquired fragment. In this paper, we employ a method using a restriction enzyme for the removal of the damaged part on the fragments. Based on the method, we found that the yield of an adapter ligated on one end is about 30%, and on both ends is about 10%, which is adequate for the amplification and sequencing.