Pseudo-Complementary DNA

伪互补DNA

• 批准号: 8072639
• 负责人: Howard Byron Gamper
• 金额: $ 19.36万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-14 至 2012-08-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072639
• 关键词: 2,6-Diaminopurine; 2-thiothymine; Address; Alleles; Applications Grants; Base Pairing; Biological Assay; Complementary DNA; DNA; DNA Ligation; DNA Resequencing; DNA biosynthesis; DNA-Directed DNA Polymerase; Detection; Development; Diagnostic; Elements; Emerging Technologies; Free Energy; Genes; Grant; Higher Order Chromatin Structure; Human; Hybrids; Length; Libraries; Ligation; Methods; Nanotechnology; Nucleic Acids; Nucleotides; Oligonucleotide Microarrays; Oligonucleotide Probes; Oligonucleotides; Performance; Pharmacogenetics; Play; Point Mutation; Polymers; Population; Preparation; Primer Extension; Property; Protocols documentation; RNA; Research; Reverse Transcription; Role; Sampling; Single Nucleotide Polymorphism; Single-Stranded DNA; Solutions; Specificity; Structure; Technology; Temperature; Testing; Thermodynamics; Time; Transcript; Work; analog; base; cost effective; design; improved; melting; nanopore; new technology; novel; novel strategies; programs; public health relevance; research study; three dimensional structure; tripolyphosphate

DESCRIPTION (provided by applicant): Conceptually, pseudo-complementary DNA (pcDNA) is a "structure-free" polymer composed of base analogs that don't interact with each other but are able to Watson-Crick pair to regular bases in a complementary probe. Base pairs in the resulting hybrids are composed of unique combinations of modified and natural nucleotides. The ability to convert native DNA or RNA into structure-free pcDNA would significantly improve the reliability and efficiency of short probes by eliminating interference from secondary structure in the target. The exquisite ability of such probes to discriminate against mismatches favors their use in the direct detection of single nucleotide polymorphisms. pcDNA targets would also facilitate the practical use of universal oligonucleotide microarrays and the development of extremely dense SNP microarrays. High throughput sequencing technologies based on oligonucleotide ligation or passage through a nanopore would also benefit. Recently, we developed a set of dNTP analogs that support enzymatic synthesis of DNA with reduced secondary structure and increased accessibility to short unmodified probes. In this grant application we propose to optimize methods for the preparation of pcDNA and to rigorously characterize the hybridization properties of this DNA with respect to accessibility, stability and specificity. Significant effort will be expended in developing a new, rapid and cost-effective approach to determining nearest neighbor free energies for both pairing and mis-pairing of novel bases such as the ones used to prepare pcDNA. This approach will use a microarray platform to simultaneously determine the melting profiles of hundreds of short duplexes. Successful completion of this research program will provide a comprehensive analysis of the practicality and benefits of pseudo-complemementary nucleic acids and establish whether microarrays can be used to simplify and accelerate the acquisition of free energy parameters for hybridization. PUBLIC HEALTH RELEVANCE: Short pieces of nucleic acid known as oligonucleotides have applications in human diagnostics, pharmacogenetics and high throughput sequencing due to their ability to interact with DNA and RNA. Unfortunately, the performance of oligonucleotides is impaired by the presence of higher order structure in naturally occurring DNA and RNA. In this grant we will evaluate a novel strategy for eliminating higher order structure in DNA thereby improving the utility of oligonucleotides for both new and existing applications. In reducing the technology to practice, we will test a new microarray based strategy for acquiring the thermodynamic parameters that describe the interaction of oligonucleotides with "structure-free" DNA. This thermodynamic library will facilitate use of the new technology.

描述（由申请人提供）：从概念上讲，伪互补DNA （pcDNA）是一种“无结构”聚合物，由碱基类似物组成，彼此不相互作用，但能够在互补探针中对规则碱基进行沃森-克里克配对。由此产生的杂合体中的碱基对由修饰的和天然的核苷酸的独特组合组成。将天然DNA或RNA转化为无结构的pcDNA的能力将通过消除靶标中二级结构的干扰，显著提高短探针的可靠性和效率。这种探针鉴别错配的精细能力有利于它们在单核苷酸多态性的直接检测中使用。pcDNA靶标也将促进通用寡核苷酸微阵列的实际应用和极密集SNP微阵列的发展。基于寡核苷酸连接或通过纳米孔的高通量测序技术也将受益。最近，我们开发了一套dNTP类似物，支持酶促DNA合成，降低了二级结构，增加了短未修饰探针的可及性。在这项拨款申请中，我们建议优化pcDNA的制备方法，并严格表征该DNA在可及性、稳定性和特异性方面的杂交特性。将花费大量的精力来开发一种新的、快速的、具有成本效益的方法，以确定用于制备pcDNA的新型碱基的配对和错配对的最近邻自由能。该方法将使用微阵列平台同时确定数百个短双工的熔化曲线。该研究计划的成功完成将提供对伪互补核酸的实用性和益处的全面分析，并确定微阵列是否可以用于简化和加速杂交自由能参数的获取。