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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

基本信息

批准号：
10112940
负责人：
Omid Veiseh
金额：
$ 46.78万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-15 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10112940
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 kinetic model magnetic beads melting milliliter models and simulation next generation sequencing novel public health relevance reference genome scale up tool

项目摘要

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)：样本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无法分析典型的1毫升人类血液样本中的所有1017个核苷酸。对DNA和RNA样品进行有效和快速的下一代测序(NGS)至关重要，即选择性捕获/保留所需的DNA基因座或序列。目前的浓缩技术(主要是多重聚合酶链式反应、杂交捕获和分子反转探针)都存在捕获一致性和捕获特异性有限的问题。第一个限制导致序列相对于另一个的较差的量化(例如，拷贝数变化)，而第二个限制导致下游的NGS读取浪费。由于大多数浓缩应用对多路复用的要求很高，由于探针和靶序列之间存在大量潜在的相互作用，因此通常很难合理地或凭经验地系统优化。PI建议开发新的杂交探针和系统，以实现对DNA和RNA序列的多路捕获和浓缩。与以前的杂交捕获技术不同，PI的方法专注于具有定制可设计的不同序列杂交动力学的探针，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。通过使用差异杂交动力学，研究小组将能够实现在平衡时无法实现的复杂的平衡前浓缩分布。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。为了进一步提高新捕获探针组设计的可预测性，该团队还将使用新的方法来快速、更准确地测量自然条件下的核酸热力学和动力学。