Millikan Sequencing by Label-Free Detection of Nucleotide Incorporation

描述（由申请人提供）：将评估无标签技术密立根测序的可行性，该技术将用于1000美元以下的哺乳动物基因组从头测序。这种新颖的合成测序方法测量了核苷酸被添加到附着在栓系头上的DNA模板上时增加的电荷。相反的电，流体动力和熵力将被用来测量头部位移，这是附着在头部的DNA长度的函数。同时检测数以百万计的珠子进行链延伸阵列将允许高通量测序。模型计算和初步结果表明，该方法可以实现准确、长读取长度和无标记的DNA测序。缺乏标签导致试剂成本可以忽略不计，而相对简单的光学导致低成本的仪器。较长的读取长度将导致较低的基因组组装成本。与454系统相比，所需的每头拷贝数要低得多，应该启用乳液PCR以外的扩增选项，例如桥式PCR，使初始样品制备更容易和更便宜。最终，该方法可用于单分子，从而进一步降低样品制备成本。拟议的为期两年的探索性项目的目标是：(1)证明使用单个系留头测序DNA的能力，以及(2)开发一种方案，允许同时检测大型头阵列以进行高通量分析。

DESCRIPTION (provided by applicant): The feasibility of a label-free technology, Millikan Sequencing, will be evaluated for de novo sequencing of mammalian genomes for under $1,000. This novel sequencing-by-synthesis approach measures the increased charge as nucleotides are added to DNA templates attached to a tethered bead. Opposing electrical, hydrodynamic and entropic forces will be used to measure the bead displacement, which is a function of the length of DNA attached to the bead. Simultaneous detection of an array of millions of beads undergoing chain elongation will allow high-throughput sequencing. Model calculations and preliminary results indicate that this method should enable accurate, long read length and label-free DNA sequencing. The lack of labels leads to negligible reagent costs while the relatively simple optics leads to a low-cost instrument. Long read lengths will result in low genome assembly cost. The much lower per-bead copy number required compared to the 454 system should enable amplification options other than emulsion PCR, such as bridge PCR, making initial sample preparation easier and cheaper. Ultimately, the method could be used on single molecules thereby further reducing sample preparation costs. The aims of the proposed two-year exploratory project are: (1) to demonstrate the ability to sequence DNA using a single tethered bead, and (2) to develop a scheme that would allow simultaneous detection of large bead arrays for high throughput analysis. PUBLIC HEALTH RELEVANCE: Nucleic acid sequence information is critical to medical research and to basic biological studies. The goal of this project is to develop a DNA sequencing system based on a label-free detection approach, Millikan Sequencing, which is capable of de novo sequencing of mammalian genomes for under $1,000. Such a platform will allow researchers and clinicians to perform the translational research ultimately required to improve human health.