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Single-cell and single-molecule laser biotechnology

机译:单细胞单分子激光生物技术

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Abstract: While lasers have found a wide field of application in the analysis of cells and biomolecules, their use in manipulation is less common. Now, new applications of lasers are emerging, which aim at cells and even molecules as biotechnological individuals: For example, in single cell gel electrophoresis individual cells are irradiated by UV laser pulses which cause radiation damage to DNA. When the whole cell is positioned in an electric field and the UV induced damages are converted into DNA strand breaks, the resulting DNA fragments are eluted out of the cell nucleus. Small fragments are running further than large ones. After staining of the DNA fragments, the cell has the appearance like a comet (therefore comet assay). The tail moment, a parameter quantifying the shape of the tail, gives information on the degree of DNA damage. The kinetics of DNA damage induction can be described by a type of exponential law with parameters which are related to radiation sensitivity of the DNA. A further emerging technique aims at DNA as a molecular individuum. One pivotal step for single molecule DNA analysis is single molecule handling. For that purpose, a DNA molecule is coupled to a micrometer sized polystyrene bead, either via an avidin-biotin bridge or, more specifically, by strand recognition, and labeled with fluorescence dyes such as DAPI. In order to visualize the dynamics of individual DNA molecules, highly sensitive video processing and single photon counting is required. Moving the polystyrene bead using optical tweezers, the molecule can be deformed, i.e., bent, turned or stretched. Using a laser microbeam, the same individual molecule can be cut into smaller portions. !14
机译:摘要:虽然激光在细胞和生物分子的分析中发现了广泛的应用领域,但在操纵中却很少使用。现在,激光的新应用正在涌现,其瞄准的是生物技术个体中的细胞甚至分子:例如,在单细胞凝胶电泳中,单个细胞被紫外线激光脉冲照射,从而对DNA造成辐射损伤。当将整个细胞置于电场中并且将紫外线诱导的损伤转化为DNA链断裂时,所得的DNA片段将从细胞核中洗脱出来。小片段比大片段运行得更快。对DNA片段染色后,细胞具有类似彗星的外观(因此进行了彗星测定)。尾巴力矩是量化尾巴形状的参数,可提供有关DNA损伤程度的信息。 DNA损伤诱导的动力学可以通过一种指数规律来描述,其参数与DNA的辐射敏感性有关。另一种新兴技术旨在将DNA作为一种分子个体。单分子DNA分析的一个关键步骤是单分子处理。为此,可通过亲和素-生物素桥或更具体地通过链识别将DNA分子与微米级的聚苯乙烯珠偶联,并用荧光染料(例如DAPI)标记。为了可视化单个DNA分子的动力学,需要高度敏感的视频处理和单光子计数。使用光镊移动聚苯乙烯珠,该分子可以变形,即弯曲,转向或拉伸。使用激光微束,可以将相同的单个分子切成较小的部分。 !14

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