Magnetic Microspheres: Synthesis And Application In Genomic DNA Extraction

With the rapid progress of life sciences and biotechnology, magnetic separation technique has become an indispensable tool because of its ease of use, rapid separation, being compatible with variety of sample matrices and amenable to automation and minimization.In this thesis, we report preparation of three types of magnetic microspheres by polymer templated method and chemical co-precipitation and their application as adsorbents for isolation of genomic DNA from three different biological samples. Polyethylene glycol (PEG) and sodium chloride were used as loading buffers. Effects of experimental parameters including composition of capturing buffer and elution time on the yields of DNA isolated using the magnetic microspheres were examined and compared with those obtained with spherical and irregular silica particles. Results show that recovery yields of DNA up to 80% can be obtained on solid-phase adsorbents with a capturing buffer composed of 20% PEG and 2 mol/L NaCl and elution time of 10 min. The magnetic microspheres are comparable with the silica particles in terms of yield and purity of DNA isolated. However, since settlement of particles by centrifugation is omitted, the protocol of DNA isolation based on magnetic microspheres allows the reduction of the sample preparation time and automation of traditionally tedious operating procedures.To understand the mechanism of DNA adsorption on the magnetic microspheres, we further study the adsorption isotherms of DNA from PEG and NaCl buffers. It was found that the adsorption of DNA on the solid-phase adsorbents can be described with Freundlich adsorption model with related coefficient above 0.994.Crushed up the calf thymus and maize kernels by whetting, Isolated the genomic DNA of calf thymus and maize kernels with magnetic microspheres after the cells were lysed by lysis buffer. We received pure DNA fragments of about 20 kb with the A260/A280 values above 1.77.The magnetic microspheres were applied to isolation of genomic DNA from wildtype Saccharomyces cerevisiae. Two different cell lysis methods were used to break up the cell walls partly and entirely, the genomic DNA fragments obtained were about 5 kb and 20 kb with the A260/A280 values in the range 1.77 to 1.89.Different magnetic microspheres, which surface modified by silane reagents,...