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Investigation of Interaction between Magnetic Silica Particles and Lambda Phage DNA fragment

Adsorption of biological macromolecules to solid surfaces has important applications among others in DNA microarrays [1], DNA-based sensors [2], high-quality DNA purification by chromatographic techniques [3] and DNA hybridization studies [4]. Nucleic acids must be isolated, purified, and concentrated from complex samples for both research and clinical diagnostic applications. Such sample preparation is commonly accomplished through solid phase extraction, relying on the reversible interactions between nucleic acids and a solid support, such as silica. The silica support required for these methods is implemented either in the form of a filter membrane [5] or as silica-coated magnetic particles [6]. Therefore detail understanding the nature of DNA interactions with silica surfaces is important. The binding between DNA and silica is based on intermolecular electrostatic interactions, dehydratation of the silica surface and DNA and intermolecular hydrogen bond between DNA and silica contact layer [7]. The mechanism of nucleic acid adsorption on the silica surface is widely discussed emphasizing the importance of presence of monovalent or divalent ions in the media [8-10]. Also the role of external parameters such as temperature, pH and/or ionic strength as driving forces need be well described [11]. Therefore, the influence of all these parameters on the DNA interactions with silica surface were investigated in this study. All key steps including DNA adsorption and desorption environment were characterized using sensitive and efficient electrochemical detection. Based on obtained results, a DNA isolation method using magnetic silica particles was suggested and successfully applied to samples of bacterial cells of Escherichia coli and Staphylococcus aureus.

DNA isolation using magnetic microparticles
For DNA isolation from PCR mixture the magnetic microparticles Dynabeads MyOne Silane (Life Technologies, Invitrogen, Norway) and magnetic stand Magnetic Particle Concentrator-S (Life Technologies, Invitrogen, Norway) were used. Experiments with magnetic particles were performed in RNA/DNA UV cleaner box UVT-S-AR (Biosan, Latvia). Multi-spin MSC-6000 centrifuge (Biosan, Latvia) placed in UV cleaner box was used for centrifuging and vortexing of a sample. For heating the Thermomixer 5355 Comfort/Compact (Eppendorf, Germany) was used.

10 µl of the MPs in the storage solution was transferred to a microcentrifuge tube and placed to a magnetic stand. The MPs were separated from solution due to an external magnetic field and the storage solution was removed. The microcentrifuge tube was then removed from magnetic stand, 20 µl of a washing solution was added to MPs and the suspension was mixed with the pipette to reach equal dispersion of the MPs in the washing solution. The microcentrifuge tube was placed to a magnetic stand and the washing solution was removed. This washing step was repeated. The composition of washing solution and the number of washing steps were optimized. Subsequently, 10 µl of the sample solution and 10 µl of the immobilization solution were added to clean MPs. The immobilization took place in the multi-spin MSC-6000 or Thermomixer 5355 Comfort/Compact depending on the need to heat the microcentrifuge tube. The settings on multi-spin MSC-6000 were as follows: 20 s of shaking and centrifugation at 2400 rpm for 1 s. Other immobilization parameters such as immobilization solution composition, presence of alkali metal chloride in immobilization solution, level of shaking, immobilization temperature and time were optimized. After the immobilization, the washing step was repeated rinsing out the immobilization solution and all potential impurities. Subsequently, 30 µl of elution solution was added to the washed MPs with adsorbed nucleic acid and the elution was carried out in the Thermomixer 5355 Comfort/Compact. Due to the heating the nucleic acids were released from the particles. The composition of elution solution and its pH, level of shaking, elution temperature and time were optimized. Using magnetic stand, the solution containing only nucleic acids was transferred into clean microcentrifuge tube and electrochemically analyzed.

Podpořeno projekty: NanoBioTECell GA CR P102/11/1068

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