Micro/Nano Magnetic Particles For Trace DNA Extraction And Selective Separation Of DNA Fragments

Functionalized magnetic particles, modified with silanol, amino or carboxyl groups, have been widely used for the separation and purification of DNA, due to their rapid magnetic separation and highly efficient DNA recovery. Effective extraction of trace DNA is of great significance for improving the detection sensitivity of nucleic acid molecule, such as the early molecular diagnosis of disease. In this thesis, we first aimed at the high detection sensitivity of nucleic acids, and investigated the extraction of trace DNA using silanol- and amino-modified magnetic silica particles as nucleic acid carriers. Secondly, with the rapid development of next generation sequencing and the discovery of free DNA in peripheral blood, the rapid preparation of desired DNA fragments is an inevitable requirement. In this context, we therefore investigated the selective separation of DNA fragments using carboxyl-coated silica magnetic particles.Three main research contents were involved in this thesis: ⅰ) Using fluorescence quantitative PCR, we measured the recovery rates of trace nucleic acids（λDNA） obtained by different types of silica magnetic particles. Then, several factors including the magnetic particle dispersibility, the oscillation of adsorption and desorption process, the elution volume, and the time of magnetic separation were analyzed to assess their effects on the DNA recovery. In addition, we also investigated the PCR amplification including silica magnetic particle-DNA complexes with the aim of improving the detection sensitivity of nucleic acid. ⅱ) Using the methods of PCR and fluorescence quantitative PCR, we studied the desorption behavior of trace DNA（λDNA） on the amino-modified silica magnetic particles by replacement of DNA with dNTP. Additionally, we also investigated the influence of amino-modified silica magnetic particles on PCR amplification and analyzed the feasibility of using amino-modified silica magnetic particles assisted PCR to improve the nucleic acid detection sensitivity. ⅲ) Under a certain salt（NaCl） concentration, we investigated the relationship between the recovered DNA obtained by carboxyl-coated silica magnetic particles and PEG concentration. Then, we developed a new method to determine the critical PEG concentration, at which DNA fragments precipitate on these particles. Further, we established a new method to determine the concentrations of NaCl and PEG for the selective separation of DNA fragments, and investigated its preliminary applications in biomedicine.Based on the mentioned above researches, some conclusions are as follows:1) The recovery of trace DNA obtained by silanol-modified silica magnetic particles was low and had a certain volatility, mainly due to the magnetic particle dispersibility. Additionally, the external factors including the oscillation of adsorption and desorption process, the elution volume, and the magnetic separation time also influenced the DNA recovery. The PCR involving silica magnetic particle-DNA complexes was found to be beneficial for improving the detection sensitivity of nucleic acid molecules.2) The efficiency of the replacement of DNA on the amino-modified silica magnetic particles with dNTP was related to the amino density, dNTP concentration, and the amount of DNA bound to the particles. Lower amino density, higher dNTP concentration and greater amount of nucleic acid bound to the particles would be of benefit to the DNA desorption.3) The recovered DNA concentration or DNA recovery exhibited the feature of a typical sigmoid. This sigmoid curve could be fitted by the logistic function (y=A₂+（A₁-A₂）/[(1+（x/x₀）^p] to determine the critical PEG concentration, at which DNA fragments could precipitated on the carboxyl-coated silica magnetic particles. By deducing the first derivative of the logistic function, the slope function of the recovery equation was achieved to develop the recovery spectrum, which could be used to determine the NaCl and PEG concentrations required for the selective DNA separation. Finally, the method mentioned above was found to be effective in its preliminary applications in biomedicine.