SBIR Phase I: Novel Method for Detecting Single Copy Genes

*9661065特诺夫斯基这个小企业创新研究第一阶段项目将探索开发一种新型的、超灵敏的原位杂交格式，用于检测琼脂糖微滴包裹的整个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目标扩增的情况下识别存在于一小部分细胞群体中的感兴趣基因。对于单拷贝基因检测，即将开发的技术将主要与基于靶扩增的方法竞争，如聚合酶链式反应。这些方法虽然敏感，但需要释放目标核酸，因为纯化的模板是扩增所必需的，使它们不适合于细胞相关核酸的检测。这种正在研究的方法将利用新型的辣根过氧化物酶化学发光底物，并结合冷却的CCD相机来检测微弱的光线。由于原位杂交不需要分离DNA，高信号密度有望检测到不同细胞位置的低DNA拷贝数。该方法可用于通过体外(转基因、基因治疗)或体内(病毒感染、转座)方法鉴定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。开发用于基因组研究和分子细胞计量学的检测微量DNA的高灵敏度分析格式，将有助于开发具有重要经济价值的材料。***

*** 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. ***