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，将应用近12种新技术来提高DNA芯片微阵列检测，定量和表征核酸的性能。这些技术包括： 一种人工扩增遗传信息系统（AEGIS），一种DNA样分子识别系统，遵循沃森-克里克规则形成双链体，但不与天然核酸相互作用。 一种自回避分子识别系统（SAMRS），其成分与天然DNA结合，但不与同一非天然SAMRS系统的其他成分结合。 同时接受AEGIS和SAMRS的DNA聚合酶和逆转录酶 可逆终止子，其终止引物延伸，但（与双脱氧核苷三磷酸终止子不同）可以在以后被移除以继续引物延伸，以及接受它们的聚合酶。这些支持“使用循环可逆终止的测序”，以在阵列上验证阵列命中。 SNAP 2技术，以16聚体的特异性但8聚体的区分能力引发DNA和RNA [Lea 06]。 使用喷墨DNA阵列合成仪进行阵列上DNA合成的新技术。 可转化的核苷酸，能够对含有非天然结构单元的DNA和RNA进行下游克隆和测序。 在夹心测定中执行以增加单个靶分析物结合到阵列的荧光标签的数量的树枝状结构。 为了验证这些技术是否可在阵列上使用，将实现以下具体目标： 1.我们将使用喷墨DNA阵列合成仪来制备结合这些技术的靶阵列，建立合成-测试-评估-重新设计-再合成的循环，这将允许创新化学品在其改善样品制备和阵列性能的能力方面进行内部基准测试。 2.我们将合成一系列支持新技术所需的化合物。 3.我们将对新技术进行基准测试，以了解其改善以下方面的能力： 3.1分析物与阵列杂交的速率与杂交的复杂性无关 测定混合物。 3.2分析物与阵列杂交的灵敏度与杂交测定混合物的复杂性无关。 3.3关于单核苷酸错配的选择性。 3.4多个阵列元件针对单个分析物的不同区域的响应均匀性。 3.5使用树枝状结构增加低丰度分析物目标的检测灵敏度。 4.然后，我们将采用创新技术，在各种样品制备架构中将AEGIS标签添加到天然样品中 5.最后，我们将开发一个系统，用于使用可逆终止子进行测序的阳性“命中”的阵列验证。公共卫生相关性这个项目，针对国家人类基因组研究所，是基于这样一个事实，即需要创新的新化学来提高基于阵列的核酸分析的性能，以满足从事基因组科学的生物医学研究人员的苛刻规格，随着微阵列性能的提高，需求也在增加。Benner实验室已经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，从样品制备到阵列分析。该提案寻求资金，以衡量这些新技术所产生的阵列性能改进，通过测试和重新设计周期进行改进，并将其提供给基因组学界。