Single DNA Detection Based On Volunme Amplification

This thesis includes three parts: Detection of single molecules and the technique of magnetic nanoparticles; Detection the single molecules of pUC19 DNA by the magnetic nanoparticles /microbeads; The quantitative determination of the single DNA associated with the anthrax lethal factor.In the chapter one of this dissertation, the detail review of detection of single molecules and the technique of magnetic nanoparticles were delivered. First of all, basic content, the characteristic of single molecules detection and several main techniques in single molecules detection were introduced. Then the application of magnetic nanoparticles was introduced. Emphases of this is the separation and detection of DNA, the immobility of enzyme, the selection of cells and the detection of immunity. In the chapter, 85 references have been referred.In the chapter two, the novel method to detect single DNA molecules was presented. The method relied on a three-component sandwich assay format. In the method, single target DNA molecules were captured by both oligonucleotide-modified magnetic nanoparticles and ～10 μm oligonucleotide-modified microbeads. First of all, we prepared the magnetic nanoparticles functionalized with oligonucleotides which were complementary to one portion of a target DNA sequence and the microbeads functionalized with oligonucleotides which were complementary to one portion of the target DNA sequence. The magnetic nanoparticles were loaded with the target DNA binding at a high ratio of magnetic nanoparticles and target DNA to ensure that only one target DNA molecule could combine to a magnetic nanoparticle. The suspension containing the target DNA molecules combined to the magnetic nanoparticles was mixed with the functionalized microbeads with a high ration of microbeads and target DNA to ensure that only one target DNA molecule with one magnetic nanoparticle could combine to a microbead. In the suspension, each target DNA molecule with one magnetic nanoparticle and one microbead. These target DNA molecules were obtained by using magnet separation. Invisible target DNA molecules could be observed through bound ～10 μm microbeads under an optical microscope.In the chapter three, we developed a method for quantitative determination of the single DNA molecules associated with the anthrax lethal factor. The yield of the hybrid was higher by increasing the time of hybridization and changing condition of vibration. We also easily took photographs of the microbeads without repeat and pretermission because we used the microcells with "coordinate". The molecular number is linear with the concentration of DNA associated with the anthrax lethal factor in the range of 1.0 × 10^-19mol/L-8.0×10^-19mol/L.