EDNA/ELCA FOR QUANTITATIVE DETERMINATION OF SPECIFIC DNA

我们描述了一种用于DNA的超灵敏量热分析，EDNA-ELCA， 它利用凝血的共同途径的扩增来产生 一种量热检测产物。 EDNA-ELCA能够检测 微量滴定板中阿摩尔量的DNA，灵敏度为200- 比常规ELISA技术高1000倍。 该分析已被 应用于PCR扩增DNA的定量检测， 用于检测突变的寡核苷酸连接测定（奥拉 与遗传性疾病镰状细胞性贫血和囊性 纤维化，以及检测kras癌基因的特异性突变 与结直肠癌有关。 我们还应用了 DNA杂交分析。 我们改进了技术， 以试纸条测定的形式呈现，其提供快速，灵敏， 无需专门设备即可获得明确结果的量热分析。 在本提案中，我们将继续开发试纸检测格式 通过进一步简化、稳定化和呈现。 我们还将 使用该技术快速和敏感的可视化特定的 电印迹DNA序列，并继续开发用于 基于微量滴定板的杂交，有或没有PCR扩增。 我们选择了三个应用程序，将展示广泛的 测定的实用性。 这些是1）检测已知的突变， 组织和粪便样品中的结肠直肠癌，2）定量和菌株 血液中丙型肝炎病毒的测定和3）人类 涂片中与宫颈癌有关的乳头状瘤病毒株， 活组织检查 这些应用程序将利用 技术，即高通量，灵敏度和简单性，而 允许该技术的不同方面的发展。 拟定的商业应用：该测定系统将允许常规 筛选DNA序列、基因突变或病毒序列特异性 对于某些疾病。该测定可以自动化和/或简化， 在一定程度上，它将适合于高通量临床 实验室或办公室。这些方法的实施可 大大提高了早期癌症检测技术的准确性。的 分析也适用于专业研究或诊断目的 研究实验室。

We describe an ultrasensitive calorimetric assay for DNA, the EDNA-ELCA, which uses amplification of the common pathway of coagulation to produce a calorimetrically detectable product. The EDNA-ELCA enables detection of attomole amounts of DNA in a microtiter plate, with a sensitivity 200- 1000 time higher than conventional ELISA techniques. The assay has been applied to quantitative detection of PCR amplified DNA, and combined with the oligonucleotide ligation assay (OLA) for the detection of mutations associated with the genetic diseases sickle cell anemia and cystic fibrosis, and the detection of specific mutations in the kras oncogene associated with colorectal cancer. We have also applied the technique to DNA hybridization assays. We have modified the technique for presentation as a dipstick assay which provides a rapid, sensitive, calorimetric assay with definitive results without specialized equipment. In this proposal we will continue to develop the dipstick assay format by further simplification, stabilization and presentation. We will also use the technology for rapid and sensitive visualization of specific electroblotted DNA sequences, and continue development of the assay for microtiter plate based hybridization, with or without PCR amplification. We have chosen three applications which will demonstrate the broad utility of the assay. These are 1) detection of known mutations in colorectal cancer in tissue and stool samples, 2) quantitation and strain determination of hepatitis C virus in blood and 3) the detection of human papilloma virus strains related to cervical cancer in smears and biopsies. These applications will make use of the strengths of the technology i.e. high throughput, sensitivity and simplicity, while allowing the development of different aspects of the technique. PROPOSED COMMERCIAL APPLICATION: This assay system would allow routine screening for DNA sequences, gen mutations or viral sequences specific for certain diseases. The assay could be automated and/or simplified to an extent where it will be suitable for a high throughput clinical laboratory or an office setting. Implementation of these approaches may greatly increase the accuracy of early cancer detection techniques. The assay is also suitable for research or diagnostic purposes in specialty research laboratories.