Force spectroscopy platform for label free genome sequencing

描述（由申请人提供）：高效、快速获取个体遗传信息的能力对预防医学的进步至关重要，对临床医学所有领域的新疗法和医疗设备的引入也至关重要。药物基因组学的新医疗保健范式将使规定的治疗与个体的基因组学特征相匹配；但是，这样的概要必须在一定的成本限制下实时提供。为了满足快速和经济的基因组测序的挑战，提出了DNA测序的一个新的基本概念：单核苷酸添加（SNA）的检测可以基于单个DNA片段的力学性质和分子结构的变化。力谱的DNA经历阻滞聚合将用于实现单分子在时间的变化分析分子力学与单核苷酸的分辨率。力谱法能够精确测定单个化学键（约0.1 nm）的端到端距离，被认为是研究分子构象变化的一种新的“分子标尺”。通过使用光学近场探针，力谱方法可以发展为具有大规模并行格式的技术，可以同时跟踪数百万个SNA的添加。该技术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。排除分离和扩增步骤进一步加快了全基因组测序的时间框架。力谱在高度平行格式的发展允许测序装置的进一步小型化和程序的自动化，通过采用微流体处理涉及SNA循环的步骤。这种并行化将在低成本的桌面设置中实现，适用于大多数生物、化学和医院实验室。力谱实验中单核苷酸敏感性的证明可以导致广泛接受该技术作为生物学研究中的基因组学工具。分子力学分析的成功示范将有助于从目前的单一医疗转向多样化的、以患者为中心的、以全基因组测定为常规测试的治疗。

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.