Force spectroscopy platform for label free genome sequencing

描述（由申请人提供）：有效和快速获得个体遗传信息的能力对于预防医学的进步以及在临床医学的所有领域引入新疗法和医疗器械至关重要。药物基因组学的新的保健范例将使规定的治疗与个体的基因组学概况相匹配;然而，这样的概况必须在真实的时间内并且在一定的成本限制内提供。为了满足提供快速和负担得起的基因组测序的挑战，提出了DNA测序的新的基本概念：可以基于单个DNA片段的机械性质和分子结构的变化来检测单个核苷酸添加（SNA）。力谱的DNA进行逮捕聚合将被用来实现一个分子在同一时间的分析，在分子力学的变化与单核苷酸的分辨率。力谱的能力，以确定一个单一的化学键（约0.1 nm）的准确度的端到端的距离被认为是一个新的“分子尺子”，研究分子构象的变化。通过使用光学近场探针，力谱方法可以发展成为具有大规模并行格式的技术，其中可以同时跟踪数百万个SNA添加。该技术将不需要标记核苷酸碱基，并且碱基识别将完全基于分子作为一个整体所经历的变化来完成。分离和扩增步骤的排除进一步加快了全基因组测序的时间范围。力谱的高度并行格式的发展允许进一步小型化的测序设备和自动化的程序，通过采用微流体涉及SNA循环的步骤。这种并行化将在低成本的桌面设置中实施，适用于大多数生物、化学和医院实验室。力谱实验中的单核苷酸灵敏度的证明可以导致该技术作为生物研究中的基因组学工具被广泛接受。分子力学分析的成功演示将有助于从目前的统一医疗转向以患者为中心的多元化治疗，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.