| The selective separation of DNA fragments with different sizes is a meaningful issue in the field of biochemistry and clinical diagnosis. For example, the selection of specific DNA size interval used as read length is one of the key steps for next-generation sequencing. Selective extraction of short DNA fragments in maternal plasma or serum has already been applied in development of risk-free methods for prenatal diagnosis of fetal genetic diseases. Gel electrophoresis is a conventional method to separate DNA fragments with different sizes on the basis of their different amounts of charges, which, however, is limited by its time consuming procedure, low recovery efficiency(<60%) and easy contamination. In this work, a facile and efficient approach has been demonstrated for size-selective separation of DNA fragments by using lysine-functionalized silica particles. At a given p H, the environmental ionic strength can be utilized to alter the electrostatic interactions of lysine-functionalized silica particles with DNA fragments and in turn the the DNA fragments on the silica particle surfaces, which exhibits a clear dependence on the DNA fragment sizes. By carefully adjusting the environmental p H and salt concentration, therefore, the use of the lysine-functionalized silica particles allows effective separation of binary and ternary DNA mixtures, for example, two different DNA fragments with sizes of 101 and 1073 bp, 101 and 745 bp, 101 and 408 bp, respectively, and three different DNA fragments with sizes of 101, 408 and 1073 bp. The following is the content of the thesis:1. We synthesize silica particles by using stober method. Lysine is attached on the surface of silica particles via silane agent. This method makes the number of amino and carboxyl groups 1:1 directly. It could avoid the drawback of controlling the ratio of amino and carboxyl groups in previous work. This material has the ability of charge reversal. Lys-Si O2 particles is positive at p H lower p I and it could adsorb DNA effectively. When p H in solution is higher than p I, DNA could be desorbed owing to their electrostatic repulsion. We obtain the high extraction efficiency of DNA.2. It is expected that the electrostatic interactions of small charged species with oppositely charged matrices decrease with the increasing of ionic strength in solution. Here, we studied the effect of the environmental ionic strength on the adsorption of DNA fragments with different sizes. In the presence of Na Cl, the electrostatic attraction between the negatively charged DNA and the positively charged particle surfaces is screened and become weaker with the ionic strength increasing, thus decreasing the DNA adsorption efficiency onto the Lys-Si O2 particles. Short DNA fragments is expected to be neutralized more easily than long ones with the Na Cl concentration increase. So we found an optimal Na Cl concentration, at which the short DNA fragments can’t adsorb onto the particle surfaces while long DNA fragments could be adsorbed. In this way, we separate different size of DNA fragments selectively. Thanks to the operation ease, the present strategy should have promising potential in practical application in size-selective DNA extraction for downstream research... |
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