Electronic Sequencing in Nanopores

描述（由申请人提供）：长期目标是一种通用仪器的纳米孔探测器芯片，能够进行廉价的从头测序，也可以用于重新测序项目。该仪器直接产生碱基依赖的电子信号，当单链基因组DNA的数千碱基长度片段依次通过电接触单壁碳纳米管探针连接的纳米孔驱动时。最终的系统旨在使用来自少于100万个细胞的DNA，从6.5倍的基因组覆盖率中提供相对高质量的序列，而无需扩增或标记。具体目的是：1)在有和无DNA分子的离子溶液中对未连接的纳米管关节纳米孔探测器进行表征，建立器件模型；2)控制未缺口纳米管关节纳米孔暴露的纳米管表面的ssDNA结合、易位和滑动；3)研究并优化DNA分子诱导的纳米管电极电导率随纳米管偏压、栅极电压和溶液性质的变化规律；4)分析并优化第一代探测器中间隙纳米管电极之间的隧道电流调制；5)设计并制作了第二代嵌入式“T”型纳米管探测器，在Kb长度的DNA链上实现了1 Kb/秒的测序；6)设计第三代纳米孔检测器，实现高通量10kb /秒/纳米孔测序。如果我们能够在每个碱基以104个碱基/秒的速度通过纳米孔时解析每个碱基，那么一个由100个这样的纳米孔阵列组成的仪器可以在大约20小时内产生一个哺乳动物基因组的高质量草图序列，每个哺乳动物基因组的成本约为1000美元。以这些降低的成本进行基因组测序将在许多方面为改善人类健康作出重要贡献，包括了解、诊断、治疗和预防疾病；环境科学与修复；而人类健康和疾病的遗传学源于对进化的理解。我们正在开发一种仪器的核心检测器，该仪器可以在大约20小时内产生一个哺乳动物基因组的高质量草图序列，每个哺乳动物基因组的成本约为1,000美元。以这些降低的成本进行基因组测序将在许多方面为改善人类健康作出重要贡献，包括了解、诊断、治疗和预防疾病；环境科学与修复；而人类健康和疾病的遗传学源于对进化的理解。

DESCRIPTION (provided by applicant): The long-term objective is a nanopore detector chip for a general utility instrument capable of inexpensive de novo sequencing that can also be used for re-sequencing projects. The instrument directly generates base-dependent electronic signals as multi-kilobase length fragments of single stranded genomic DNA is driven sequentially through nanopores articulated with electrically contacted single walled carbon nanotube probes. The final system is intended to provide a relatively high quality sequence from =6.5-fold coverage of a genome using DNA from fewer than 1 million cells, with no amplification or labeling. The specific aims are: 1) Characterize ungapped nanotube articulated nanopore detectors in ionic solution with and without DNA molecules to establish a device model; 2) Control ssDNA binding, translocation, and sliding on the nanotube surface exposed in ungapped nanotube articulated nanopores; 3) Study and optimize DNA molecule induced field effect modulation of nanotube electrode conductance in ungapped nanotube articulated nanopores as a function of nanotube bias, gate voltage, and solution properties; 4) Analyze and optimize tunneling current modulations between gapped nanotube electrodes in the first generation detector; 5) Design and fabricate a second generation detector with embedded 'T' nanotube geometry and achieve 1 Kb/sec sequencing on Kb length strands of DNA; 6) Design a third generation nanopore detector for high throughput 10 Kb/sec/nanopore sequencing. If we are able to resolve each base as it passes through a nanopore at the rate of 104 bases/sec as proposed here, an instrument with an array of 100 such nanopores could produce a high quality draft sequence of one mammalian genome in ~20 hours at a cost of approximately $1,000/mammalian genome. Genomic sequencing at these reduced costs would make vital contributions to improved human health on many fronts, including the understanding, diagnosis, treatment, and prevention of disease; environmental science and remediation; and the genetics of human health and disease derived from the understanding of evolution. PROJECT HEALTH RELEVANCE We are developing the core detector of an instrument that could produce a high-quality draft sequence of one mammalian genome in ~20 hours at a cost of approximately $1,000/mammalian genome. Genomic sequencing at these reduced costs would make vital contributions to improved human health on many fronts, including the understanding, diagnosis, treatment, and prevention of disease; environmental science and remediation; and the genetics of human health and disease derived from the understanding of evolution.