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。更具体地说，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。当核苷酸被整合到生长的DNA聚合物中时，能量从聚合酶内被激发的供体荧光团转移到核苷酸内的受体荧光团，刺激碱基特异性整合信号的发射，直接实时检测。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，结合起来创建这个新的测序系统。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，成本约为1000美元，并允许充分的过采样产生冗余数据，从而将错误降到最低。该系统正在开发中，以识别病原体（或其变异）并使全面的基因组分析成为可能。

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.