Sequencing by transcription using single-molecule field-effect transistors

描述(申请人提供)：拟议研究的长期目标是设计和建造一种DNA测序系统，该系统可以以低于100美元的价格对整个人类基因组进行测序，包括样本制备，并且该系统的产品成本远低于5,000美元。根据拟议研究开发的系统在成本上至少比招标的目标高出一个数量级，同时保持所有业绩指标。在这方面，它将打破通往基因组医学的障碍。整个系统是基于在转录或聚合样品DNA的同时，通过连接到每个纳米管上的酶的运动，在碳纳米管CMOS阵列上对DNA进行测序的。酶在每个转录步骤的运动都会转化为电导率的变化。当其中一个核苷酸的浓度比其他三个核苷酸的浓度降低时，将通过一系列更快的步骤检测到停顿。所有核苷酸之间的迭代将导致解码彼此之间的所有碱基位置。这个测试已经成功地在另一个基于光学的系统中进行了测序，但读取长度有限，仅为5kb。目前的系统可以实现50kb的读取长度。初步数据显示，酶可以单分子方式负载在纳米管上。此外，其他研究小组已经证明，在纳米管上可以监测酶的运动。虽然这是一个很好的开始，但第一阶段的工作将研究酶与纳米管的最佳结合，并展示使用Eve Biomedical的分析进行测序；调查这种测序方法的准确性、读取长度和吞吐量限制。如果成功，主要目标是在第一阶段结束时使用原型系统完成微生物DNA(大肠杆菌)的测序，从而完全基准拟议的体系结构。在第一阶段之后，将建造一个低成本的工作台系统来执行整个人类基因组的测序，目标成本被定义为这次赠款募集的目标。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is the design and construction of a DNA sequencing system that can sequence the whole human genome for under $100 including sample preparation and with the cost of goods of the system well under $5,000. The system developed under the proposed research is at least an order of magnitude in cost better than the target of the solicitation while maintaining all performance metrics. In that respect it will break the barrier towards genomic medicine. The overall system is based on sequencing DNA on a carbon nanotube CMOS array via motion of enzymes attached to each nanotube while transcribing or polymerizing the sample DNA. The enzyme motion at each transcription step translates to conductivity changes. When one of the nucleotides is reduced in concentration compared to the other three a pause will be detected in a series of faster steps. Iterating between all nucleotides will lead to decoding of all base positions with respect to each other. This assay has successfully sequenced in an alternate optical-based system, but with limited read length at the 5kb order. The current system can achieve >50kb read length. Preliminary data shows that enzymes can be loaded at a single molecule fashion on a nanotube. Furthermore, other groups have shown that motion of enzymes can be monitored while on nanotubes. While this is a great start, Phase I work will investigate the most optimal attachment of enzymes to nanotubes and show sequencing using Eve Biomedical's assay; investigating accuracy, read length and throughput limits of this sequencing approach. If successful, the main goal is to completely sequence a microbial DNA (Ecoli) at the end of Phase I using a prototype system, and thus completely benchmark the proposed architecture. Beyond Phase I, a low cost bench top system will be constructed to perform sequencing of Whole Human Genome with the target cost defined as goal of this grant solicitation.