Adsorption Of DNA By Modified Fe₃O₄Magnetic Microspheres

Extraction of nucleic acids from biological samples is one of the basic techniques ofmolecular biology and clinical medicine in the field of life science research. The nucleic acidextraction methods include phenol-chloroform extraction, CTAB separation method,bromide ethidium （EtBr）-cesium chloride （CsCl） the different gradient centrifugation,salting-out method, a solid phase extraction method and so on. These methods are oftencomplex, expensive, time-consuming and poor purity of the samples. The surface modifiedmagnetic particles as a carrier were applicated in the solid phase separation, the magneticmaterials are easy to separation at a magnetic field outer, are widely used to separate andpurify DNA. The separation technology is a fast and effective method of separation, and alsocan avoid the organic reagent contamination. The prospects for development is very well.Using four different programs to prepare of magnetic microspheres, with PMIDA, aminoacids, vinyl phosphonic acid and polyethylene glycol as modification reagent, a novel batchof biosorbents was prepared. The modified microspheres can be applicated to find theoptimum adsorption conditions and used to separate and purify DNA from fresh corn. Themain contents are as flow:1. The two magnetic microspheres (MNP-PMIDA-Zn²⁺and MNP-PMIDA-Cu²⁺)wereprepared with PMIDA and Zn²⁺,Cu²⁺as modification reagent and chelating reagent,respectively. The Fourier transform infrared spectrohotometry was used to characterize themicrospheres. The modification content on the surface of magnetic microspheres wasdetermined by determining the supernatant concentration of the PMIDA. The effecting factorson the DNA adsorption were studied. The maximum adsorption capacity ofMNP-PMIDA-Zn²⁺and MNP-PMIDA-Cu²⁺were21.78mg·g^-1,17.31mg·g^-1, respectively. Theadsorption isotherms of two adsorbents were fitted with Langmuir and Freundlich model, theywere the monolayer and multimolecular layer adsorption, respectively. At the ammoniaconcentration of3.5%and4.5%, respectively, the DNA was eluted from a surface of theadsorbents. The separation and purification of DNA were achieved. The modifiedmicrospheres can be applicated to extract DNA from fresh corn. This method is very simpleand fast. DNA can be extracted from the sample successfully, with higher extraction efficiency and higher purity DNA（A260/A280:1.6382and1.2927）.2. The modified with Lysine, Arginine, and histidine-tag magnetic Fe₃O₄microsphereswere used to adsorb DNA. The microspheres were characterized with Fourier transforminfrared spectrohotometry and TEM. The adsorption conditions were optimized. The resultsshowed that the pH is at5.0, ionic strength （NaCl concentration） is2.0mol L^-1, temperaturekeeps at room temperature, the contact time is for20min,25min and25min, respectively, theadsorption efficiency are the highest. The adsorption isotherms were fitted with the Langmuirand Freundlich model. For Lysine and Arginine modified magnetic microspheres,theprocesses belong to the monolayer adsorption, the adsorption process of histidine-tagmodified magnetic microspheres are the monolayer adsorption and adding multimolecularlayer adsorption. The modified microspheres can be applicated to extract DNA from freshcorn, the extraction purity （A260/A280） were higher than1.8. DNA was extracted from thefresh corn sample successfully with higher extraction efficiency, higher purity DNA andobvious bands in the electrophoretic patterns. The results indicated that DNA was extractedsuccessfully by the amino acids modified magnetic microspheres.3. Polymer coated magnetic microspheres were prepared with vinyl phosphonic acid,and then were chelated with Zr4+to study the separation and purification of DNA.The Fouriertransform infrared spectrohotometry and TEM were used to characterize the microspheres.The results showed that the pH is at5.0, ionic strength （NaCl concentration） is2.0mol L^-1,temperature keeps at room temperature,the contact time is for20min, the adsorptionefficiency are the highest （75.43%）. The adsorption isotherms were fitted with the Langmuirand Freundlich model, The results indicated that the adsorption processes were found to agreebetter the Freundlich model with multimolecular layer adsorption. When the PEGconcentration is25%, the DNA elution ratio is up to87.53%, so as to achieve the separationand purification of DNA. The modified microspheres can be applicated successfully to extractDNA from fresh corn, with higher extraction efficiency, higher purity of DNA（A260/A280=1.9900） and obvious bands in the electrophoretic patterns. The results showed that DNA wasextracted successfully by using the polymer modified magnetic microspheres.4. PEG8000modified magnetic Fe₃O₄microspheres were prepared and used to adsorbDNA. The orthogonal experiments were was used to find the best preparation conditions. The microspheres were characterized with FTIR.When the solution acidity is at PH6.0, ionicstrength （NaCl concentration） is1.5mol L^-1, temperature keeps at30℃, contact time is for15min, the adsorption efficiency are the highest. The adsorption isotherms were fitted withthe Langmuir and Freundlich model, the adsorption process of PEG modified magneticmicrospheres is a multimolecular layer adsorption. The modified microspheres can beapplicated successfully to extract DNA from fresh corn, with higher extraction efficiency,higher purity（A260/A280=1.9289） of DNA and obvious bands in the electrophoretic patterns.The results showed that DNA was extracted successfully by using the magnetic microspheres.