br Experimental br Results and discussions br Conclusion br

Experimental
Results and discussions
Conclusion
Introduction The joining of DNA fragments using DBU is an essential process in gene cloning. One of the important parameters for performing DNA ligation efficiently is the temperature [1]. In the case of DNA strands with cohesive ends, ligation is generally performed at 12–16°C since higher temperatures may reduce the ligation efficiency by melting annealed DNA ends [1], [2], [3]. The ligation of blunt-ended DNA is usually carried out at room temperature with a high concentration of T4 DNA ligase [1], [4]. The optimal temperature of commonly used DNA ligase is around 37°C and therefore the above ligation conditions are not optimal for the action of DNA ligase. Several attempts to increase the ligation efficiency by improving thermal conditions for the reaction have been reported. Lund, Duch and Pedersen [2] reported that a high enzyme activity and DNA annealing were balanced by constant temperature cycling, as a result of which the ligation efficiency was increased. Ranjan and Rajagopal [3] demonstrated that DNA fragments with 2-bp overhangs were ligated using T4 DNA ligase at room temperature with high efficiency when the DNA fragments were subjected to heating followed by flash freezing prior to the ligation reaction. It was recently reported that an enzyme immobilized on ferromagnetic particles was heated and activated under a radio frequency alternating magnetic field due to heat dissipation from the particles caused by magnetic hysteresis and eddy currents with no influence on the activity of free enzyme around the particles [5]. This suggests that DNA ligase can be activated utilizing the heat generation of ferromagnetic particles subjected to an ac magnetic field with little effect on the annealing of DNA ends. In this article, we carry out the ligation of DNA fragments with cohesive ends using T4 DNA ligase immobilized on ferromagnetic particles and show that the ligation efficiency is increased by applying an ac magnetic field. The basic concept of our method is illustrated in Fig. 1. DNA ligase is immobilized on the surface of ferromagnetic particles and a reaction solution containing the DNA ligase/ferromagnetic particle hybrids and DNA fragments to be ligated is set at low temperature suitable for the annealing of DNA ends. When a radio frequency alternating magnetic field is applied, the ferromagnetic particles generate heat caused by magnetic hysteresis and eddy currents [6], [7], [8], [9], [10], [11] and DNA ligase on the particles is selectively heated up and activated. If we optimize the experimental conditions, the activation of DNA ligase can be carried out almost without melting annealed DNA ends. The present method is so simple that it can easily be combined with other methods for efficient DNA ligation including the addition of molecular crowding agents such as polyethylene glycol [12], [13], [14] or hexamine cobalt chloride [15].
Materials and method