High Throughput Sequencing Using Single Molecule Millikan Sequencing

描述(由申请人提供)：将评估用于哺乳动物基因组从头测序的单分子密立根测序的可行性，成本低于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系链珠子上的单个DNA模板上时增加的电荷，而不需要任何标记。相反的电动力、流体动力和恢复力会导致珠子运动，这是附着在珠子上的DNA长度的函数。同时光学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，而相对简单的暗场光学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。单模板方法消除了乳胶或桥式聚合酶链式反应的需要，使样品制备变得更容易和更便宜。拟议的为期两年的探索性项目的目的是通过监测不断增长的DNA链的内在电荷的变化来证明对单个DNA模板进行测序的可行性。 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。该项目的目标是证明基于无标记检测方法的DNA测序系统的可行性，单分子密立根测序可能能够以低于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 single molecule Millikan Sequencing for de novo sequencing of mammalian genomes for under $1,000 will be evaluated. This novel sequencing-by-synthesis approach measures the increased charge as nucleotides are added to a single DNA template attached to a tethered bead without any need for labels. Opposing electrical, hydrodynamic and restoring forces result in bead motion that is a function of the length of DNA attached to the bead. Simultaneous optical 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 dark-field optics leads to a low-cost instrument. Long read lengths will result in low genome assembly cost. The single template approach eliminates the need for emulsion or bridge PCR, making sample preparation much easier and cheaper. The aim of the proposed two-year exploratory project is to demonstrate the feasibility of sequencing a single DNA template by monitoring changes in the intrinsic charge of a growing DNA chain. PUBLIC HEALTH RELEVANCE: Nucleic acid sequence information is critical to medical research and to basic biological studies. The goal of this project is to demonstrate the feasibility of DNA sequencing system based on a label-free detection approach, single molecule Millikan Sequencing, which may be 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.