Millikan Sequencing by Label-Free Detection of Nucleotide Incorporation

描述（由申请人提供）：将评估无标记技术密立根测序的可行性，用于哺乳动物基因组的从头测序，费用低于1000美元。 这种新的合成测序方法测量了当核苷酸被添加到附着在拴系珠上的DNA模板时增加的电荷。 相反的电、流体动力和熵力将用于测量珠位移，其是附着于珠的DNA长度的函数。 同时检测经历链延长的数百万个珠的阵列将允许高通量测序。 模型计算和初步结果表明，这种方法应该能够实现准确的，长的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，而相对简单的光学器件导致低成本仪器。长读段长度将导致低基因组组装成本。 与454系统相比，所需的每个微珠的拷贝数低得多，这应该能够实现除乳液PCR之外的扩增选择，例如桥式PCR，使得初始样品制备更容易且更便宜。 最终，该方法可以用于单分子，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）证明使用单个拴系珠进行DNA测序的能力，以及（2）开发一种允许同时检测大珠阵列以进行高通量分析的方案。 公共卫生相关性：核酸序列信息对医学研究和基础生物学研究至关重要。 该项目的目标是开发一种基于无标记检测方法的DNA测序系统，即密立根测序，该系统能够以低于1000美元的价格对哺乳动物基因组进行从头测序。 这样一个平台将使研究人员和临床医生能够进行最终改善人类健康所需的转化研究。

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.