OBJECTIVETo develop a solid phase PCR method by covalent single point immobilization for recycle utilization of human genome.

METHODSPolymethacrylamide gel was selected as a solid PCR carrier based on DNA-hydrogel copolymer chemistry presented by Mirzabekov. (CH2)6NH2 amino-modified PCR product and randomly fractured formic acid-modified plasmid pGEM-T-HLA-G were used as templates. The specificity of the attachment chemistry was characterized by acrylamide gel electrophoresis, and the thermal stability of method was demonstrated by PCR. This method was applied for the recycle utilization of human genome. Sequencing was used to exclude the possibility of introduced mutations during modification and immobilization procedures.

RESULTSThe PCR detections of plasmid DNA and human genome DNA immobilized by polymethacrylamide gel was successful. The thermal stability of method was successfully demonstrated by performing PCR after 16 rounds of standard 36 PCR cycles. And the sequencing was found no mutation.

CONCLUSIONThe DNA immobilization method with polymethacrylamide gel as a solid phase carrier is stable and specific, which can be a possible approach for realizing recycle utilization of human genome for whole-genome sequencing and SNP detection.

Electrophoresis, Polyacrylamide Gel ; Genome, Human ; Humans ; Hydrogels ; Immobilized Nucleic Acids ; analysis ; Polymerase Chain Reaction ; methods

OBJECTIVETo Prepare surface functional magnetic microspheres for the separation of vascular endothelial growth factor (VEGF) nucleic acid and lactase enzyme immobilization.

METHODSUsing suspension polymerization methods to copolymerize MA-styrene containing magnetite nanoparticles and GMA-styrene also containing magnetite nanoparticles, respectively. Both the carboxyl-modified magnetic microspheres and epoxy-modified magnetic microspheres were obtained. In addition, the chloromethyl-modified magnetic microspheres were prepared by seedy microemulsion. The magnetic microspheres bound with b-gamma IgG were determined by radioimmunoassay (RIA), and the separation of VEGF nucleic acid and lactase enzyme immobilization were performed by carboxyl-modified magnetic microspheres.

RESULTSTransmission electron microscopy (TEM), energy-dispersive spectroscopy (EDS) and infrared (IR) spectra showed that the products of polymer magnetic microspheres were monodispersed and that the magnetic particles were uniformly distributed in the microsphere with special functional group on the surface of the microsphere. RIA showed that three kinds of magnetic microspheres could be bound with b-gamma IgG and the absorption of b-gamma IgG reached 75 micrograms/mg, especially for the carboxyl-modified magnetic microspheres. The carboxyl-modified magnetic microspheres can be used for the separation of VEGF nucleic acid by coupling with corresponding primer. Moreover, the immobilized enzyme was proportional to the amount of the carboxyl-modified magnetic microspheres.

CONCLUSIONSThe surface functional magnetic polymer microspheres can be bound with active bio-substance, and have a wide application prospect in the fields of biology and medicine.

Adsorption ; Endothelial Growth Factors ; chemistry ; Enzymes, Immobilized ; Immunoglobulin G ; Lactase ; metabolism ; Magnetics ; Microspheres ; Nanotechnology ; Nucleic Acids ; isolation & purification ; Particle Size

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 microM. 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.
Chimera ; DNA ; Gene Expression ; Genetic Engineering ; Immobilization ; Immobilized Nucleic Acids ; Ligation ; Mercaptoethanol ; Molecular Biology ; Multiplex Polymerase Chain Reaction ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; Polymerization ; Polymers ; Reaction Time ; Surface Properties