Real-time DNA Sequencing

描述（由申请人提供）：VisiGen Biotechnologies，Inc.正在开发一种测序系统，其中聚合酶和核苷酸一起作为DNA碱基身份的直接分子传感器。更具体地，该技术检测修饰的核苷酸和荧光修饰的聚合酶之间的相互作用。当核苷酸被掺入到生长的DNA聚合物中时，能量从聚合酶内的激发的供体荧光团转移到核苷酸内的受体荧光团，刺激实时直接检测的碱基特异性掺入特征的发射。尖端技术，包括单分子检测，荧光分子化学，计算生物化学和生物分子工程，相结合，创造这种新的测序系统。DNA样本将在大规模并行阵列中进行处理，这将使大型基因组在不到一天的时间内测序，费用约为1000美元，并允许足够的过采样以产生冗余数据，从而最大限度地减少错误。该系统正在开发，以识别病原体（或其变体），并实现全面的基因组分析。 证明了VisiGen技术的原理：使用固定化的供体标记的聚合酶和2种不同的受体标记的dNTP，在单分子水平上实时检测序列信息。鉴于我们联邦资助的努力取得的成功，VisiGen系统的进一步发展，以实时测序单个DNA分子的阵列显然是必要的。拟议研究的具体目标是改进测序化学，设计、构建和测试下一代单分子DNA测序仪器。这台名为Alpha-1的仪器将被放置在VisiGen，并加速技术开发。该仪器将由贝勒医学院、人类基因组测序中心（休斯顿，德克萨斯州）和SeqWright公司的研究人员用于beta测试。（德克萨斯州休斯顿）。参与这些beta测试的人员的反馈将被纳入下一阶段的技术开发。

DESCRIPTION (provided by applicant): VisiGen Biotechnologies, Inc. is developing a sequencing system in which polymerase and nucleotides act together as direct molecular sensors of DNA base identity. More specifically, the technology detects the interaction between a modified nucleotide and a fluorescently-modified polymerase. As a nucleotide is incorporated into the growing DNA polymer, energy transfers from an excited donor fluorophore within the polymerase to an acceptor fluorophore within the nucleotide, stimulating the emission of a base-specific incorporation signature that is directly detected in real-time. Cutting-edge technologies, including single molecule detection, fluorescent molecule chemistry, computational biochemistry, and biomolecule engineering, are combined to create this novel sequencing system. DNA samples will be processed in massively parallel arrays that will enable large genomes to be sequenced in less than a day for approximately $1000 and permit sufficient oversampling to produce redundant data that will minimize errors. The system is being developed to identify pathogens (or variations thereof) and to enable comprehensive genome analysis. Proof of principle for VisiGen's technology is demonstrated: sequence information has been detected at the single molecule level in real-time using an immobilized, donor-labeled polymerase and 2 different acceptor-labeled dNTPs. Given the success of our federally-funded efforts, further development of the VisiGen system to sequence arrays of single DNA molecules in real-time is clearly warranted. The specific aims of the proposed research are to refine the sequencing chemistry and to design, build and test the next generation single-molecule DNA sequencing instrument. This instrument, Alpha-1, will be placed at VisiGen and accelerate technology development. This instrument will be used for beta testing by researchers at the Baylor College of Medicine, Human Genome Sequencing Center (Houston, TX) and SeqWright, Inc. (Houston, TX). Feedback from personnel involved in these beta tests will be incorporated into the next phase of technology development.