Millikan Sequencing by Label-Free Detection of Nucleotide Incorporation

描述(由申请人提供)：将评估一种无标记技术--密立根测序技术的可行性，用于哺乳动物基因组的从头测序，费用低于1,000美元。这种新的合成测序方法测量了当核苷酸添加到连接到拴系珠上的DNA模板上时增加的电荷。相反的电动力、流体动力和熵力将被用来测量珠子的位移，这是附着在珠子上的DNA长度的函数。同时检测正在进行链延长的数百万个珠子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、长阅读长度和无标记的DNA测序。没有标签导致试剂成本可以忽略不计，而相对简单的光学元件导致了一种低成本的仪器。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，所需的每粒拷贝数低得多，这应该会使乳胶聚合酶链式反应以外的扩增方法成为可能，例如桥式聚合酶链式反应，从而使初始样品制备更容易、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)展示使用单个系链珠子对DNA进行测序的能力，以及(2)开发一种方案，允许同时检测用于高通量分析的大型珠粒阵列。 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，它能够以低于1,000美元的价格对哺乳动物基因组进行从头测序。这样的平台将允许研究人员和临床医生进行最终需要的转化性研究，以改善人类健康。

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.