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  • br Materials and method br Results

    2021-03-05


    Materials and method
    Results and discussion We carried out DNA ligation using free and immobilized T4 DNA ligase in the absence of a magnetic field at 16°C, where the enzyme concentration (final concentration after mixing the solutions for the reaction) in both cases was 0.40μg/mL, and found that the total amounts of ligated DNA in the cases of free and immobilized T4 DNA ligase were, respectively, 75 ± 11 and 82 ± 4 fmol. This suggests that DNA ligase was still active after having been immobilized on the ferromagnetic particles although the ligation efficiency was slightly decreased compared to that in the case of free DNA ligase. The surface of iron particles used in the present study was not modified with any molecules and the particles were negatively charged in water. Therefore it is supposed that some domains in T4 DNA ligase were positively charged and DNA ligase molecules were attached to the particles by electrostatic force as in the case of the attachment of α-amylase, chitinase and lipase to magnetic particles [5], [16], [17]. We next carried out DNA ligation with free T4 DNA ligase under an ac magnetic field of 0.34MHz and confirmed that there was no appreciable effect of the magnetic field on the ligation efficiency even in the case of the maximal field amplitude in the present study (30kA/m). The dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles in the absence of a magnetic field on the ambient temperature is shown in Fig. 3, where the ordinate axis represents the ligation efficiency normalized by that at 16°C. Note that the total amount of DNA ligated at 16°C was 75 fmol. The ligation efficiency reached maximum in the temperature range of 12–16°C. The activity of T4 DNA ligase increases with an increase in the temperature up to its optimal temperature (37°C). However, higher temperatures dissociate DNA fragments joined by CID 2745687 pairing at their overhanging ends, which decreases the ligation efficiency. The dependence of the ligation efficiency on the temperature shown in Fig. 3 is the result of competition between the above two factors. When the ac magnetic field was applied, DNA ligase immobilized on the ferromagnetic particles was selectively heated caused by heat dissipation from the particles and therefore the ligation efficiency increased. Fig. 4 shows the dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.34MHz at 16°C on the amplitude of the magnetic field, where the ordinate axis represents the ligation efficiency normalized by that in the absence of a magnetic field. Note that the total amount of DNA ligated in the absence of a magnetic field was 75 fmol. The ligation efficiency was increased with an increase in the field amplitude. In the absence of a magnetic field, the efficiency of DNA ligation using DNA ligase immobilized on the ferromagnetic particles was slightly lower than that using free DNA ligase as mentioned above. However, the rate of increase in the efficiency of DNA ligation using immobilized DNA ligase in the presence of the ac magnetic field was much higher than the rate of decrease in the efficiency caused by the immobilization. In our previous study [5], we investigated the heat generation from the same ferromagnetic iron particles under an ac magnetic field and it CID 2745687 is estimated that the average surface temperature of the particles in the present case increases from 20 to 27°C as the field amplitude is increased from 15 to 30kA/m. The inset in Fig. 4 shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles. In summary, we carried out the ligation of DNA fragments with cohesive ends using T4 DNA ligase 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.