Chemistry to Improve Human Genomic Array Analysis Architectures

描述(申请人提供)：该项目面向国家人类基因组研究所，将应用近12项新技术来提高DNA芯片微阵列的检测、定量和表征核酸的性能。这些技术包括： 人工扩展遗传信息系统(Aegis)，一种类似DNA的分子识别系统，遵循沃森-克里克规则形成双链，但不与天然核酸相互作用。 一种自回避分子识别系统(SAMRS)，其成分确实与天然DNA结合，但不与同一非天然SAMRS系统的其他成分结合。 同时接受宙斯盾和SAMRS的DNA聚合酶和逆转录酶 可逆终止子，终止引物延伸，但(与双脱氧核苷三磷酸终止子不同)可在以后移除以继续引物延伸，以及接受它们的聚合酶。它们支持“使用循环可逆终止进行排序”，以便在数组命中仍在数组上时验证数组命中。 SNAP2技术，它以16聚体的特异性启动DNA和RNA，但具有8聚体的区分力[Lea06]。 使用喷墨DNA阵列合成器进行在线DNA合成的新技术。 可转换的核苷酸，能够对含有非天然构建块的DNA和RNA进行下游克隆和测序。 树枝状结构在夹心分析中执行，以增加单个目标分析物结合到阵列上的荧光标签的数量。 为了验证这些技术是否可在阵列上使用，将实现以下特定目标： 1.我们将使用喷墨DNA阵列合成器来制备结合了这些技术的目标阵列，建立一个合成-测试-评估-重新设计-再合成的循环，使内部能够对创新的化学物质进行内部基准测试，以改善样品制备和阵列性能。 2.我们将合成一系列支持新技术的化合物。 3.我们将根据新技术的改进能力对其进行基准测试： 3.1与杂交化验混合物的复杂性无关的分析物与阵列的杂交率。 3.2分析物对阵列杂交的灵敏度与杂交分析混合物的复杂程度无关。 3.3单核苷酸错配的选择性。 3.4针对单个分析物的不同区域的多个阵列元件的响应的一致性。 3.5检测低丰度分析物目标的灵敏度，使用树枝状结构提高。 4.然后，我们将采用创新技术，在各种样品制备架构中为天然样品贴上宙斯盾标签 5.最后，我们将开发一个系统，使用可逆终止子进行测序，以对阳性“命中”进行在线验证。公共卫生相关性这个项目针对国家人类基因组研究所，前提是需要创新的新化学方法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要求，这些要求随着微阵列性能的提高而增加。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一步，从样品准备到阵列分析。这项提案寻求资金，以对这些新技术产生的阵列性能改进进行基准测试，通过测试和重新设计的周期来改进它们，并将它们提供给基因组学社区。

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.