FLUORESCENCE CORRELATION SPECTROSCOPY IN CELLS W/ TWO PHOTON EXCITATION

具有两个光子激发的细胞荧光相关光谱

• 批准号: 6349390
• 负责人: PETRA SCHWILLE
• 金额: $ 0.31万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349390
• 关键词: bioengineering /biomedical engineering; biological products; biomedical equipment development; biomedical resource; genome; lasers; technology /technique

In view of the utilization of human genome data the capability of sequencing the genetic information of single cells without prior amplification would potentiate speed and sensitivity of gene function recognition. We propose to carry out single molecule sequencing by following the stepwise addition of base pairs during production of the complementary DNA strand of a given (unknown) template strand by the enzyme DNA polymerase. The incorporation of each base at the polymerase active site is to be recognized by detection of a specific fluorescent marker. Preliminary studies on DNA synthesis on immobilized template strands utilized the fluorescent dye PicoGreen (Molecular Probes Inc.), and it is shown that individual molecules of newly synthesized DNA can be detected. Next, we attempt to detect the separate binding events of PicoGreen molecules to the newly generated DNA with a fluorescence correlation spectroscopy setup. The DNA sequencing approach will also utilize nanofabricated compon ents for DNA manipulation and possible fluid flow.

鉴于人类基因组数据的利用， 测序单细胞的遗传信息，而无需事先 扩增将提高基因功能的速度和灵敏度 识别. 我们建议进行单分子测序， 在生产过程中逐步添加碱基对后， 在一个实施例中，通过将给定（未知）模板链的互补DNA链与给定（未知）模板链的互补DNA链连接， DNA聚合酶。 每个基地的合并， 聚合酶活性位点是通过检测特异性 荧光标记。 细胞DNA合成的初步研究 固定化的模板链利用荧光染料PicoGreen （分子探针公司），研究表明， 可以检测到新合成的DNA。 接下来，我们尝试检测 PicoGreen分子与新产生的 DNA与荧光相关光谱设置。 的DNA 测序方法还将利用纳米制造的组件， DNA操作和可能的液体流动。