Pseudo-Complementary DNA
伪互补DNA
基本信息
- 批准号:7871949
- 负责人:
- 金额:$ 23.2万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2010
- 资助国家:美国
- 起止时间:2010-05-14 至 2012-02-29
- 项目状态:已结题
- 来源:
- 关键词:2,6-Diaminopurine2-thiothymineAddressAllelesApplications GrantsBase PairingBiological AssayComplementary DNADNADNA LigationDNA ResequencingDNA biosynthesisDNA-Directed DNA PolymeraseDetectionDevelopmentDiagnosticElementsEmerging TechnologiesFree EnergyGenesGrantHigher Order Chromatin StructureHumanHybridsLengthLibrariesLigationMethodsNanotechnologyNucleic AcidsNucleotidesOligonucleotide MicroarraysOligonucleotide ProbesOligonucleotidesPerformancePharmacogeneticsPlayPoint MutationPolymersPopulationPreparationPrimer ExtensionPropertyProtocols documentationRNAResearchReverse TranscriptionRoleSamplingSingle Nucleotide PolymorphismSingle-Stranded DNASolutionsSpecificityStructureTechnologyTemperatureTestingThermodynamicsTimeTranscriptWorkanalogbasecostcost effectivedesignimprovedmeltingnanoporenew technologynovelnovel strategiesprogramspublic health relevanceresearch studythree dimensional structuretripolyphosphate
项目摘要
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,假互补DNA(pcDNA)是一种“无结构”聚合物,由彼此不相互作用但能够与互补探针中的常规碱基Watson-Crick配对的碱基类似物组成。所得杂交体中的碱基对由修饰的核苷酸和天然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中二级结构的干扰来显著提高短探针的可靠性和效率。这种探针区分错配的精确能力有利于它们在单核苷酸多态性的直接检测中的使用。pcDNA靶还将促进通用寡核苷酸微阵列的实际使用和极密集SNP微阵列的开发。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,我们开发了一组dNTP类似物,其支持具有减少的二级结构和增加的对短的未修饰探针的可及性的DNA的酶促合成。在此授权申请中,我们建议优化制备pcDNA的方法,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。大量的努力将花费在开发一种新的,快速的和具有成本效益的方法,以确定最近邻自由能的配对和错配的新的碱基,如用于制备pcDNA。该方法将使用微阵列平台来同时确定数百个短双链体的解链概况。这项研究计划的成功完成将提供一个全面的分析的实用性和伪互补核酸的好处,并建立微阵列是否可以用来简化和加速杂交自由能参数的获取。
公共卫生相关性:被称为寡核苷酸的短核酸片段由于其与DNA和RNA相互作用的能力而在人类诊断学、药物遗传学和高通量测序中具有应用。不幸的是,寡核苷酸的性能受到天然存在的DNA和RNA中存在的高级结构的损害。在这项资助中,我们将评估一种消除DNA中高阶结构的新策略,从而提高寡核苷酸在新的和现有应用中的效用。在减少技术实践中,我们将测试一种新的基于微阵列的策略,用于获取描述寡核苷酸与“无结构”DNA相互作用的热力学参数。这个热力学库将促进新技术的使用。
项目成果
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