Enrichment of DNA/RNA Sequences based on Pre-equilibrium Hybridization Kinetics

 描述（由申请人提供）： 样品中核酸分子的序列和浓度包含大量的科学和临床信息，可用于了解途径和指导治疗。然而，我们目前用于核酸序列分析的工具在分析典型的1 mL人血液样品内的所有1017个DNA核苷酸方面达不到几个数量级。富集，即所需DNA基因座或序列的选择性捕获/保留，对于DNA和RNA样品的有效和快速的下一代测序（NGS）至关重要。目前的富集技术（主要是多重PCR、杂交捕获和分子倒置探针）都受到捕获均匀性有限和捕获特异性有限的影响。第一限制导致序列相对于彼此的不良定量（例如拷贝数变异），并且第二限制导致下游浪费的NGS读段。由于大多数富集应用的高多重要求，由于探针和靶序列之间的大量潜在相互作用，通常难以合理地或凭经验地系统优化。PI建议开发新型杂交探针和系统，以允许DNA和RNA序列的多重捕获和富集。与以前的杂交捕获技术不同，PI的方法专注于具有与不同序列杂交的定制可设计动力学的探针，并寻求利用精确的预测理解来设计在特定时间点产生所需序列捕获行为的探针。通过使用差分杂交动力学，研究小组将能够实现在平衡时无法实现的复杂的预平衡富集分布。研究团队将使用一种独特的知识驱动的设计过程，基于核酸的生物物理模型，并仅使用最小的经验优化。为了进一步增强新捕获探针组设计的可预测性，该团队还将使用新方法快速，更准确地测量天然条件下的核酸热力学和动力学。

 DESCRIPTION (provided by applicant): The sequences and concentrations of nucleic acid molecules within a sample hold vast amounts of scientific as well as clinical information that can be used to understand pathways and inform treatment. However, our current tools for nucleic acid sequence analysis fall orders of magnitude short of analyzing all 1017 nucleotides of DNA within a typical 1 mL sample of human blood. Enrichment, i.e. the selective capture/retention of desired DNA loci or sequences, is crucial to effective and rapid next-generation sequencing (NGS) of DNA and RNA samples. Current enrichment techniques (predominantly multiplexed PCR, hybrid capture, and molecular inversion probes) all suffer from limited uniformity of capture and limited capture specificity. Th first limitation results in poor quantitation of sequences relative to one another (e.g. copy number variations), and the second limitation results downstream in wasted NGS reads. Due to the high multiplexing requirement of most enrichment applications, it is generally difficult to systematically optimize either rationally or empirically, due to the large number of potential interactions between probes and target sequences. The PI proposes to develop novel hybridization probes and systems to allow multiplexed capture and enrichment of DNA and RNA sequences. Unlike previous hybrid capture techniques, the PI's approach focuses on probes with custom designable kinetics of hybridization to different sequences, and seeks to utilize precise predictive understanding to design probes that produce desired sequence capture behavior at particular points in time. By using differential hybridization kinetics, the research team will be able to achieve complex pre-equilibrium enrichment distributions that cannot be achieved at equilibrium. The research team will use a uniquely knowledge-driven design process, based on biophysical models of nucleic acids, and use only minimal empirical optimization. To further enhance the predictability of new capture probe set design, the team will also use novel methods to quickly and more accurately measure nucleic acid thermodynamics and kinetics at native conditions.