Chemistry to Improve Human Genomic Array Analysis Architectures

改善人类基因组阵列分析架构的化学

• 批准号: 7618559
• 负责人: STEVEN A BENNER
• 金额: $ 48万
• 依托单位: FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7618559
• 关键词: Adopted; Architecture; Benchmarking; Binding; Biological Assay; Chemicals; Chemistry; Cloning; Communities; DNA; DNA Microarray Chip; DNA Sequence; DNA biosynthesis; DNA-Directed DNA Polymerase; Detection; Dideoxynucleosides; Elements; Fluorescence; Funding; Genetic; Genomics; Housing; Human; Individual; Information Systems; Ink; Laboratories; Messenger RNA; National Human Genome Research Institute; Nucleic Acids; Nucleotides; Oligonucleotides; Performance; Philosophy; Poly(A) Tail; Polymerase; Preparation; Primer Extension; Process; RNA; RNA-Directed DNA Polymerase; Research Personnel; Sampling; Science; Specificity; Structure; System; Technology; Testing; Transcript; Transferase; Validation; base; improved; innovation; innovative technologies; instrument; meetings; molecular recognition; new technology; nucleobase; nucleoside triphosphate; public health relevance; response; tripolyphosphate

DESCRIPTION (provided by applicant): This project, targeted for the National Human Genome Research Institute, will apply nearly a dozen new technologies to improve the performance of DNA chip microarrays as they detect, quantitate, and characterize nucleic acids. The technologies include: An artificially expanded genetic information system (AEGIS), a DNA-like molecular recognition system that forms duplexes following Watson-Crick rules, but does not interact with natural nucleic acids. A self-avoiding molecular recognition system (SAMRS), whose components do bind to natural DNA but do not bind to other components of the same unnatural SAMRS system. DNA polymerases and reverse transcriptases that accept both AEGIS and SAMRS Reversible terminators, which terminates primer extension but (unlike dideoxynucleoside triphosphate terminators) can be later removed to continue primer extension, and polymerases that accept them. These support "sequencing using cyclic reversible termination" to validate array hits while they are still on the-array. SNAP2 technology that primes DNA and RNA with the specificity of a 16mer but the discriminatory power of 8mers [Lea06]. Novel technology for on-array DNA synthesis using an inkjet DNA array synthesizer. Convertible nucleotides that enable downstream cloning and sequencing of DNA and RNA containing unnatural building blocks. Dendrimeric structures that perform in sandwich assays to increase the number of fluorescent tags bound to an array by an individual target analyte. To validate these technologies for on-array use, the following specific aims will be met: 1. We will use the ink jet DNA array synthesizer to prepare target arrays incorporating these technologies, establishing a cycle of synthesis-test-evaluate-redesign-resynthesis that will allow in-house benchmarking of the innovative chemistries with respect to their ability to improve sample preparation and array performance. 2. We will synthesize a range of compounds needed to support the new technologies. 3. We will benchmark the new technologies with respect to their ability to improve: 3.1 Rates of hybridization of analytes to arrays independent of the complexity of the hybridization assay mixture. 3.2 Sensitivity of hybridization of analytes to arrays independent of the complexity of the hybridization assay mixture. 3.3 Selectivity with respect to single nucleotide mismatches. 3.4 Uniformity of response of multiple array elements targeted against different regions of a single analyte. 3.5 Sensitivity of detection of low abundance analyte targets, increased using dendrimeric structures. 4. We will then adopt innovative technologies to append AEGIS tags to natural samples in various sample preparation architectures 5. Last, we will develop a system for on-array validation of positive "hits" using sequencing using reversible terminators. PUBLIC HEALTH RELEVANCE This project, targeted for the National Human Genome Research Institute, is premised on the fact that innovative new chemistries are needed to improve the performance of array-based analysis of nucleic acids to meet the demanding specifications of biomedical researchers who do genomic science, demands that have increased as the performance of microarrays has improved. The Benner laboratory has invented approximately a dozen new chemical technologies that can be applied to each step of the genomic analysis, from sample preparation to array analysis. This proposal seeks funds to benchmark the improvements in array performance generated by these new technologies, improve them by a cycle of test and redesign, and provide them to the genomics community.

描述（由申请人提供）：该项目针对国家人类基因组研究所的针对性，将应用近十二种新技术来提高DNA芯片微阵列的性能，以检测，定量和表征核酸。这些技术包括： 人为扩展的遗传信息系统（AEGIS），一种类似DNA的分子识别系统，遵循Watson-Crick规则形成双链体，但不与天然核酸相互作用。 一个自我避免的分子识别系统（SAMR），其成分确实与天然DNA结合，但不与同一非自然SAMRS系统的其他组件结合。 DNA聚合酶和逆转录酶都接受宙斯盾和SAMR 可逆的终止剂，终止底漆延伸，但（与二乙氧基核苷不同三磷酸终止剂不同）可以稍后去除以继续启动延伸，以及接受它们的聚合酶。这些支持“使用循环可逆终端进行测序”以验证阵列仍在阵列上时击中。 SNAP2技术的DNA和RNA具有16mer的特异性，但具有8mers的歧视能力[LEA06]。 使用喷墨DNA阵列合成器进行阵列DNA合成的新技术。 可转换的核苷酸，可以使DNA和RNA的下游克隆和测序含有不自然的构建块。 在三明治测定中执行的树突聚合物结构增加了单个目标分析物与阵列结合的荧光标签的数量。 为了验证这些技术以进行阵列使用，将满足以下特定目标： 1。我们将使用Ink Jet DNA阵列合成器来制备结合这些技术的目标阵列，从而建立合成检验 - 审核 - 评估 - 雷德斯尼格 - 重新分辨率的周期，该周期将允许对创新化学的内部基准测试，以提高样品制备和阵列性能。 2。我们将综合支持新技术所需的一系列化合物。 3。我们将根据新技术的提高能力进行基准测试： 3.1分析物与阵列的杂交速率与杂交测定混合物的复杂性无关。 3.2分析物对阵列杂交的敏感性与杂交测定混合物的复杂性无关。 3.3相对于单核苷酸不匹配的选择性。 3.4针对单个分析物不同区域的多个阵列元件的响应均匀性。 3.5检测低丰度分析物靶标的灵敏度，使用树状大分子结构增加。 4。然后，我们将采用创新技术将aegis标签附加到各种样品制备体系结构中的自然样品 5。最后，我们将使用可逆终结器使用测序来开发一个用于在阵列验证正面“命中”的系统。公共卫生相关性是针对国家人类基因组研究所的针对性的，其前提是需要创新的新化学物质来改善基于阵列的核酸分析的性能，以满足从事基因组科学的苛刻规格，这些研究人员的要求随着微型流量的提高而增加了基因组科学的要求。本纳实验室发明了大约十几种新的化学技术，可以应用于基因组分析的每个步骤，从样本制备到阵列分析。该提案寻求资金来基准这些新技术产生的阵列性能的改进，通过测试和重新设计的周期来改进它们，并将其提供给基因组学界。