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T7 RNA polymerase engineering and RNA amplification

T7 RNA 聚合酶工程和 RNA 扩增

基本信息

批准号：
7502612
负责人：
Andrew D Ellington
金额：
$ 67.66万
依托单位：
ACCACIA INTERNATIONAL, INC.
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7502612
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

项目摘要

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.

微阵列系统在全球基因表达谱分析中的广泛应用正在引领生物学研究，