EDNA/ELCA FOR QUANTITATIVE DETERMINATION OF SPECIFIC DNA

我们描述了一种超灵敏的DNA量热分析方法，EDNA-ELCA， 它利用凝血的共同途径的放大来产生 一种量热法可检测到的产品。EDNA-ELCA可实现检测 在微量滴定板中的阿托摩尔量的DNA，灵敏度为200- 比常规的酶联免疫吸附试验高1000倍。化验结果已经被 应用于定量检测PCR扩增的DNA，并与 检测突变的寡核苷酸连接试验(OLA) 与遗传性疾病有关的镰状细胞性贫血和囊性贫血 纤维化和KRAS癌基因特异性突变的检测 与结直肠癌有关。我们还应用了这项技术 进行DNA杂交检测。我们已经对技术进行了修改 以试纸试纸的形式呈现，提供快速、灵敏、 量热分析，结果明确，不需要专门的设备。 在这项提案中，我们将继续开发试纸条分析格式 通过进一步简化、稳定和列报。我们还将 使用该技术快速而灵敏地可视化特定的 电印迹DNA序列，并继续发展该方法 以微量平板为基础的杂交，有或没有聚合酶链式反应扩增。 我们选择了三个应用程序，它们将展示广泛的 该检测方法的实用性。这些是1)检测到的已知突变 组织和粪便标本中的结直肠癌，2)定量和菌株 血液中丙型肝炎病毒的检测和3)人的检测 涂片和涂片中与宫颈癌相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，即高吞吐量、灵敏度和简单性，而 允许该技术的不同方面的发展。 拟议的商业应用：该检测系统将允许常规 筛选DNA序列、基因突变或病毒序列特异性 治疗某些疾病。该分析可以自动化和/或简化为 适用于高吞吐量临床应用的范围 实验室或办公室环境。实施这些方法可以 大大提高了早期癌症检测技术的准确性。这个 化验也适用于专业的研究或诊断目的 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.