Force spectroscopy platform for label free genome sequencing

描述(由申请人提供)：有效和快速地获得个人遗传信息的能力对于促进预防医学的进步以及在临床医学的所有领域引入新的疗法和医疗设备至关重要。药物基因组学的新保健范例将使处方治疗与个体的基因组学特征相匹配；然而，这种特征必须实时提供，并在一定的成本限制内。为了应对快速和负担得起的基因组测序的挑战，提出了一种新的DNA测序的基本概念：单核苷酸加成(SNA)的检测可以基于单个DNA片段的机械性质和分子结构的变化。DNA受阻聚合的力谱将被用来实现单核苷酸分辨率的分子力学变化的一次一个分子的分析。作用力光谱学能够以单个化学键(约0.1 nm)的精度确定末端到末端的距离，被认为是研究分子构象变化的一种新的“分子尺子”。通过使用光学近场探头，力光谱方法可以改进为具有大规模并行格式的技术，其中可以同时跟踪数百万SNA的添加。这项技术将不需要标记核苷酸碱基，碱基调用将完全基于整个分子所经历的变化。不包括分离和扩增步骤，进一步加快了整个基因组测序的时间框架。高度平行格式的力谱学的发展使测序设备进一步小型化，并通过在涉及SNA循环的步骤中使用微流体实现了程序的自动化。这种并行化将在适合大多数生物、化学和医院实验室适应的低成本桌面设置中实施。在力谱学实验中证明单核苷酸的敏感性可以导致该技术作为生物学研究中的基因组学工具被广泛接受。分子力学分析的成功示范将有助于从目前的统一医疗转向以患者为中心的多样化治疗，并将全基因组测定作为常规测试。

DESCRIPTION (provided by applicant): Ability to obtain genetic information of individuals efficiently and rapidly is critical to the advancement of preventive medicine, as well as to introduction of new therapies and medical devices in all areas of clinical medicine. The new healthcare paradigm of pharmacogenomics will match the prescribed treatment to the genomics profile of the individual; however, such a profile must be made available in real time and within certain cost constraints. To meet the challenge of delivering fast and affordable genome sequencing, a new basic concept for DNA sequencing is proposed: detection of a single nucleotide addition (SNA) can be based on changes in mechanical properties and molecular structure of single DNA fragments. Force spectroscopy of DNA undergoing arrested polymerization will be used to implement one-molecule-at-the-time analysis of changes in molecular mechanics with a single nucleotide resolution. The ability of force spectroscopy to determine the end-to-end distance with the accuracy of a single chemical bond (approximately 0.1 nm) is recognized as a new "molecular ruler" with which to study changes in molecular conformation. By using optical near field probes, the methods of force spectroscopy can be advanced into techniques having massively parallel format, where millions of SNA additions can be followed at the same time. The technique will not require labeling of nucleotide bases, and base calling will be done exclusively on the basis of changes experienced by the molecule as a whole. Exclusion of separation and amplification steps further speeds up the timeframe of the whole genome sequencing. The development of force spectroscopy in the highly parallel format allows for further miniaturization of the sequencing device and automation of the procedures by employing microfluidics for steps involving SNA cycle. This parallelization will be implemented in a low cost table-top setup suitable for adaptation in a majority of biological, chemical, and hospital laboratories. Demonstration of single nucleotide sensitivity in force spectroscopy experiments can lead to a wide acceptance of the technique as a genomics tool in biological research. Successful demonstration of molecular mechanics assay will help to move from the present day uniform medical treatment to a diversified, patient-centered treatment with determination of a full genome as a routine test.