Surface Polarity Dependent Solid-state Molecular Biological Manipulation with Immobilized DNA on a Gold Surface
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As the demand for large-scale analysis of gene expression using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We compared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM)of mercaptohexanol or hepthanethiol, which create hydrophilic or hydrophobic surface properties, respectively. A hydrophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA molecules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The optimal DNA concentration for immobilization was found to be 0.5 M. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized conditions,molecular biology techniques including DNA hybridization,ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We demonstrated from our present analysis the importance of surface polarity in solid-phase molecular biological applications. A hydrophilic SAM generated a far more favorable environment than hydrophobic SAM for solid‐state molecular techniques. Our findings suggest that the conditions and methods identified here could be used for DNA‐DNA hybridization applications such as DNA chips and for the further development of solid-phase genetic engineering applications that involve DNA-enzyme interactions.
Abstract
As the demand for large-scale analysis of gene expression using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We compared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM)of mercaptohexanol or hepthanethiol, which create hydrophilic or hydrophobic surface properties, respectively. A hydrophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA molecules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The optimal DNA concentration for immobilization was found to be 0.5 M. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized conditions,molecular biology techniques including DNA hybridization,ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We demonstrated from our present analysis the importance of surface polarity in solid-phase molecular biological applications. A hydrophilic SAM generated a far more favorable environment than hydrophobic SAM for solid‐state molecular techniques. Our findings suggest that the conditions and methods identified here could be used for DNA‐DNA hybridization applications such as DNA chips and for the further development of solid-phase genetic engineering applications that involve DNA-enzyme interactions.
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