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

基于预平衡杂交动力学的 DNA/RNA 序列富集

• 批准号: 9243282
• 负责人: David Yu Zhang
• 金额: $ 46.78万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-15 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9243282
• 关键词: Acids; Algorithms; Arts; Base Sequence; Behavior; Binding; Bioinformatics; Biological; Blood; Buffers; Chemistry; Clinical; Complex; Copy Number Polymorphism; Custom; DNA; DNA Sequence; DNA sequencing; Data; Disease; Early Diagnosis; Equilibrium; Gene Expression; Human; Human Genome; Hybrids; Inherited; Kinetics; Knowledge; Measurement; Measures; Methods; Modeling; Molecular; Nucleic Acid Hybridization; Nucleic Acids; Nucleotides; Oligonucleotides; Organism; Pathway interactions; Process; Protocols documentation; RNA; RNA Sequences; Reagent; Research; Running; Sampling; Sequence Analysis; Side; Silent Mutation; Specificity; Speed; Surface; System; Techniques; Technology; Temperature; Thermodynamics; Time; Variant; base; biophysical analysis; biophysical model; design; disease diagnosis; falls; human DNA; instrument; intercellular communication; interest; magnetic beads; melting; milliliter; models and simulation; next generation sequencing; novel; public health relevance; reference genome; scale up; tool

 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.

 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科学和临床信息，可用于了解途径并为治疗提供信息。然而，我们目前的核酸序列分析工具还无法分析典型 1 mL 人类血液样本中所有 1017 个 DNA 核苷酸。富集，即选择性捕获/保留所需的 DNA 基因座或序列，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、杂交捕获和分子倒转探针）都受到捕获均匀性和捕获特异性有限的影响。第一个限制导致序列相对于彼此的定量较差（例如拷贝数变异），第二个限制导致下游 NGS 读取浪费。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存在大量潜在的相互作用，通常很难理性或凭经验系统地优化。 PI 提议开发新型杂交探针和系统，以实现 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 的方法侧重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。通过使用微分杂交动力学，研究小组将能够实现平衡时无法实现的复杂的预平衡富集分布。研究团队将使用基于核酸生物物理模型的独特的知识驱动设计流程，并且仅使用最少的经验优化。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在自然条件下快速、更准确地测量核酸热力学和动力学。