In summary we carried out the ligation

In summary, we carried out the ligation of DNA fragments with cohesive ends using T4 vinorelbine receptor immobilized on ferromagnetic particles and found that the ligation efficiency was increased under a radio frequency alternating magnetic field caused by heat generation from the particles. In the present method, DNA ligase is immobilized on ferromagnetic particles and therefore, if DNA ligation is carried out under moderate temperature conditions, DNA ligase on particles after the reaction may be recovered using a magnet and reused. Note that the inactivation temperature of T4 DNA ligase has been experimentally estimated to be 38°C, where the inactivation temperature is defined as the temperature at which the concentration of active enzyme becomes half of the total enzyme concentration [18]. In the present case, although T4 DNA ligase immobilized on the ferromagnetic particles was heated under the ac magnetic field, the particle\'s surface temperature, ranging from 20 to 27°C, was much lower than the above inactivation temperature since the ambient temperature was as low as 16°C. We will be analyzing the reusability of T4 DNA ligase immobilized on ferromagnetic particles in detail. Furthermore, magnetic particles can be manipulated using an alternating magnetic field or a gradient magnetic field without any difficulty [19], [20], [21], [22], [23]. So if we use DNA ligase/ferromagnetic particle hybrids, DNA ligation can be carried out at a specific position in micro reactors or micro total analysis systems (μ-TASs). In the case of blunt-end ligation or the ligation of DNA fragments with 2-bp overhangs, the optimal experimental conditions may be different from those in the present case, that is, the ligation of DNA fragments with 4-bp overhangs. We will be systematically investigating the dependence of the ligation efficiency on the experimental parameters such as the amplitude and the frequency of the ac magnetic field, the number density of particles, and the ambient temperature for various types of ligations. We believe that our ligation method could be useful for efficient cell transformation. We will also be analyzing the transformation efficiency of recombinant DNA prepared with the present ligation method.
Acknowledgements This work was supported by a Grant for the Program for the Strategic Research Foundation at Private Universities S1101017 organized by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan and JSPS KAKENHI Grant Numbers JP22560209 and JP5K05842.
There is an increased need for new antibiotics because many bacterial pathogens are developing resistance to existing drugs due to overuse and overprescription of these drugs. The problem is magnified by the lack of new antibacterial agents and the reduced interest of the pharmaceutical industry to invest in their discovery and development. There is a more significant need for treatments in the Gram-negative arena as there are less options for treatment and target inhibition is more difficult. One strategy for overcoming resistance to existing antibacterial drugs is the development of drugs that inhibit new or novel targets. Bacterial NAD-dependent DNA ligase (LigA) was selected as a new target because of its essentiality for growth of a broad spectrum of Gram-positive and Gram-negative pathogens and limited homology to the human DNA ligases, which use ATP as a substrate instead of NAD. DNA ligases are critical in DNA repair, replication, and recombination.