Oxford Academic. Mogens Duch. Cite Cite Anders H. Select Format Select format. Permissions Icon Permissions. Table 1. Open in new tab Download slide. Search ADS. Issue Section:. Download all slides. Comments 0. Add comment Close comment form modal. I agree to the terms and conditions. You must accept the terms and conditions.
Add comment Cancel. Submit a comment. Comment title. You have entered an invalid code. Submit Cancel. Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion. The inset in Fig. 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.
The standard deviations are obtained from 6 independent experiments. Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.
The ordinate axis represents the ligation efficiency under an ac magnetic field, which is normalized by that in the absence of a magnetic field.
The inset shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles, noting that the surface temperature increases with an increase in the field amplitude.
Indeed, no appreciable increase in the temperature of the reaction solution was detected since the volume fraction of the heat-dissipating ferromagnetic particles in the solution final volume fraction after mixing the solutions for the reaction was as low as 3. We analyzed heat transfer between ferromagnetic particles and the solvent fluid surrounding the particles in order to understand the thermal conditions in the present experiment in more detail.
Since the volume fraction of the particles was very low in the present experiment, the particles can be assumed to be thermally isolated from each other. The steady-state temperature distribution around a spherical particle obtained by solving the heat conduction equation is:. The volume fraction of regions around particles, the temperature of which is higher than a certain threshold, T c , obtained from Eq.
However, in the present case, phosphodiester bonds between DNA fragments are formed by DNA ligase immobilized on the surface of magnetic particles and therefore annealed DNA fragments have to travel through the higher temperature region to the particle's surfaces see Fig.
When the surface temperature of magnetic particles in the former case is equal to the ambient temperature in the latter case, the activities of DNA ligase in those cases are equal to each other since DNA ligase is immobilized on the surface of magnetic particles. Comparing the ligation efficiency under the ac magnetic field as a function of the particle's surface temperature inset in Fig. Therefore the difference between the ligation efficiencies in those cases increased with an increase in the temperature.
The present result clearly shows that DNA ligase immobilized on ferromagnetic particles is activated by heating with an ac magnetic field having much less effect on the annealing of DNA ends than the case of uniform heating.
If the field amplitude is increased further, although the activity of DNA ligase may increase, the effect of heat dissipation from ferromagnetic particles on DNA annealing may become more significant at the same time and therefore, it is supposed that the ligation efficiency may reach the maximum at a higher field amplitude.
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.
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].
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. National Center for Biotechnology Information , U. Journal List Biochem Biophys Rep v. Biochem Biophys Rep.
Published online Oct Author information Article notes Copyright and License information Disclaimer. Hisao Morimoto: pj. This article has been cited by other articles in PMC. Associated Data Supplementary Materials Supplementary material. Abstract We present a simple method for efficient DNA ligation utilizing the heat generation of ferromagnetic particles subjected to an ac magnetic field. Open in a separate window.
Materials and method 2. DNA ligation with T4 DNA ligase immobilized on ferromagnetic particles We carried out the ligation of and bp DNA fragments with cohesive ends using T4 DNA ligase immobilized on ferromagnetic particles, where the ligation occurred through the cohesive ends created by the digestion since the other ends had no phosphate group as mentioned in the previous section. Appendix A.
Transparency document Supplementary material Click here to view. Supplementary material Click here to view. Appendix B. Supplementary material Supplementary material Click here to view.
References 1. Green J. Marcel Dekker, Inc. This article explains the basics of DNA ligation. DNA ligase EC 6. In molecular biology it is commonly used for the insertion of restriction enzyme-generated DNA fragments into vector backbones. The DNA ligation reaction itself has two basic steps. Firstly the DNA ends have to collide by chance and stay together long enough for the ligase to join them. This is the most inefficient part of the reaction, but is easier at low temperatures.
Well, as you will probably know, all molecules move faster at higher temperatures so you can imagine that it is going to be easier for two DNA ends to collide and stay together if they are gently floating through the solution at low temperature, rather than whizzing about as they would be at higher temperatures. For cohesive ends, there is an additional reason; lower temperatures stabilize the hydrogen bonding between the complementary nucleotides, which really helps to keep things in place.
The second step is the enzymatic reaction, which is shown schematically in Figure However, the enzyme will work very slowly at this temperature so a long e. Originally published on 31 October ; updated and republished on 5 December
0コメント