T7 RNA polymerase engineering and RNA amplification

T7 RNA 聚合酶工程和 RNA 扩增

• 批准号: 7108369
• 负责人: Andrew D Ellington
• 金额: $ 67.21万
• 依托单位: ACCACIA INTERNATIONAL, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7108369
• 关键词: Affect; Area; Attention; Bacteriophages; Biotin; Cells; Classification; Complementary DNA; Condition; Consensus; DNA; DNA Ligases; DNA Sequence; DNA-Directed DNA Polymerase; Development; Diagnostic; Engineering; Enzyme Stability; Enzymes; Gene Expression; Gene Expression Profiling; Genes; Genetic Transcription; Goals; Grant; Hela Cells; Human; Hyperactive behavior; In Vitro; Label; Laboratories; Length; Literature; Measures; Messenger RNA; Methods; Microarray Analysis; Molecular Profiling; Mutagenesis; Nucleotides; Numbers; Performance; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Physicians; Play; Population; Procedures; Process; Property; Protocols documentation; Publishing; RNA; RNA amplification; RNA chemical synthesis; RNA-Directed DNA Polymerase; Range; Rate; Reaction; Recovery; Reproducibility; Research Personnel; Ribonuclease H; Role; Sampling; Screening procedure; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Standardization; Standards of Weights and Measures; Structure; Surrogate Markers; System; T7 RNA polymerase; Temperature; Testing; Time; Transcript; Transcription Initiation Site; Validation; Variant; Work; base; biological research; design; directed evolution; enzyme substrate; improved; insight; interest; mutant; novel; prognostic; programs; promoter; rapid technique; research study; thermostability; vaccine development

DESCRIPTION (provided by applicant): Expanding use of microarray systems for global gene expression profiling is leading biological research, providing diagnostic and prognostic value to physicians and facilitating target discovery during drug and vaccine development. There is strong interest in extending this microarray capability to more RNA-limited material to the point that a single cell can be profiled with confidence. The most widely used method for preparing samples for microarray analysis, which uses cDNA synthesis from mRNA followed by T7 RNA polymerase-based amplification, currently lacks the necessary sensitivity to meet these emerging needs. To overcome these methodological limitations, we propose to increase the final amplified RNA yield by 1) improving the efficiency of T7 RNA polymerase using structure-based engineering with directed evolution of function, 2) by optimizing the design of the promoter-template construct for specific amplification needs and 3) by re-standardization of cDNA synthesis for lower RNA inputs. The combined improvements are expected to lower the cell-limited RNA sample requirement by 100-fold compared to current, commercially available kits. This will enable microarray gene analysis with as little as 1 ng of total RNA with a single round of amplification or microarray profiling from a single cell (-10 pg of total RNA) after two rounds of amplification.

描述（由申请人提供）：将微阵列系统用于全球基因表达谱分析是领先的生物学研究，为医生提供诊断和预后价值，并促进药物和疫苗开发期间的靶点发现。人们对将这种微阵列能力扩展到更多RNA限制的材料，以使单个细胞可以有信心地进行分析，有着强烈的兴趣。用于制备用于微阵列分析的样品的最广泛使用的方法，其使用从mRNA合成cDNA，然后基于T7 RNA聚合酶的扩增，目前缺乏满足这些新兴需求的必要灵敏度。为了克服这些方法学上的局限性，我们建议通过以下方式来提高最终扩增的RNA产量：1）使用具有功能定向进化的基于结构的工程化来提高T7 RNA聚合酶的效率，2）通过针对特定扩增需求优化启动子-模板构建体的设计，以及3）通过针对较低RNA输入重新标准化cDNA合成。与目前市售的试剂盒相比，这些综合改进预计将细胞限制的RNA样品需求降低100倍。这将使微阵列基因分析能够在单轮扩增中使用少至Ing的总RNA，或在两轮扩增后从单个细胞（约IOpg的总RNA）进行微阵列分析。