SBIR Phase I: Novel Method for Detecting Single Copy Genes

SBIR 第一阶段：检测单拷贝基因的新方法

• 批准号: 9661065
• 负责人: Jan Trnovsky
• 金额: $ 7.5万
• 依托单位: ONE CELL SYSTEMS, INC
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9661065&HistoricalAwards=false
• 关键词: SBIR; Phase; Novel; Method; Detecting

*** 9661065 Trnovsky This Small Business Innovation Research Phase I project will explore development of a novel, ultrasensitive in situ hybridization format for detecting single copy genes in whole cells or nuclei encapsulated in agarose microdrops. A major goal of this research is to identify genes of interest present in a small fraction of the cell population without using target amplification. For single copy gene detection, the technology to be developed will compete primarily with methods based on target amplification, such as PCR. Although sensitive, these methods require release of target nucleic acids because purified templates are necessary for amplifications, making them unsuitable for cell-associated nucleic acid detection. This method to be researched will utilize novel chemiluminescent substrates for horseradish peroxidase in combination with a cooled CCD camera for detecting low light levels. Because no isolation of DNA is needed for in situ hybridizations, high signal densities are expected to permit detection of low DNA copy numbers at different cellular locations. The method can be used for identifying genes transferred to cellular genomes by in vitro (transfection, gene therapies) or in vivo (viral infections, transposition) methods and determining the percentage of cells containing transferred genes. Development of high sensitivity assay formats for detecting minute amounts of DNA for genomics research and molecular cytometics will contribute to development of economically important materials. ***

* 9661065 Trnovsky 这个小企业创新研究第一阶段项目将探索开发一种新的，超灵敏的原位杂交形式，用于检测封装在琼脂糖微滴中的全细胞或细胞核中的单拷贝基因。 这项研究的一个主要目标是在不使用靶扩增的情况下鉴定存在于一小部分细胞群体中的感兴趣的基因。 对于单拷贝基因检测，待开发的技术将主要与基于靶扩增的方法（如PCR）竞争。 尽管敏感，但这些方法需要释放靶核酸，因为纯化的模板对于扩增是必需的，使得它们不适合于细胞相关核酸检测。 该方法将利用新型辣根过氧化物酶荧光底物与冷却CCD相机相结合，用于检测弱光水平。 因为原位杂交不需要分离DNA，所以预期高信号密度允许在不同细胞位置检测低DNA拷贝数。 该方法可用于鉴定通过体外（转染、基因治疗）或体内（病毒感染、转座）方法转移到细胞基因组的基因，并测定含有转移基因的细胞的百分比。 开发用于检测基因组学研究和分子细胞学的微量DNA的高灵敏度测定格式将有助于开发经济上重要的材料。 ***