第二代测序技术——新一代基因组测序技术原理及应用.pptVIP

Optical Detection System光学探测系统 光谱分离 激发光源 全内反射荧光 ZERO-MODE WAVEGUIDEs 零模波导for Single-Molecule Analysis at High Concentrations M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, W. W. WebbCornell University, Clark Hall, Ithaca, NY 14853,?USA Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of ZERO-MODE WAVEGUIDEs consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of ZERO-MODE WAVEGUIDEs for performing single-molecule experiments at high concentrations. Science 31 January 2003: 682  Pacific Biosciences Zero Mode Waveguide零模波导 Pacific Biosciences’s SMRT chip Each chip contains thousands of ZMWs. Each ZMW is a cylindrical hole tens of nanometers in diameter, perforating a thin metal (e.g. aluminum) film supported by a transparent substrate 激发光源 When the ZMW is illuminated through the transparent substrate by laser light, the wavelength of the light is too large to pass through the waveguide’s aperture. Attenuated evanescent light from the excitation beam decays exponentially and penetrates the lower 20-30nm of each waveguide, creating a detection volume of only 20 zeptoliters (10-21 liters). This dramatic reduction in the detection volume provides the needed 1000-fold improvement in rejection of background fluorescence. 隐失波 ? λ ZMW with Anchored Polymerase Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal claddi