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NOVEL ULTRASENSITIVE COLORIMETRIC ASSAY FOR DNA

新型超灵敏 DNA 比色测定

基本信息

批准号：
3493560
负责人：
MARK X TRISCOTT
金额：
$ 5万
依托单位：
AMPLISTAR, INC.
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-09-01 至 1994-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3493560
关键词：
DNA binding proteins biotin blood coagulation clotting factor colorimetry fibrinogen gene mutation human subject method development polymerase chain reaction reptile poison thrombin

项目摘要

The detection of specific DNA sequences for the diagnosis of genetic diseases, cancer, and infectious disease is becoming increasingly widespread. A number of innovative techniques have been employed, including the oligonucleotide ligation assay and reverse dot-blot hybridization. The end-point, and to a certain extent the effectiveness of these assays is determined by the sensitivity of the DNA detection techniques used. Progress has been significantly enhanced through the use of the polymerase chain reaction (PCR). However, implementation of PCR can be expensive, as well as equipment and expertise intensive. We have applied a technique with proven effectiveness in the field of immunoassays for the non-isotopic detection of DNA; initial data indicates an increase in sensitivity 10(2)-10(3) times higher than conventional techniques. The assay, which we have called EDNA-ELCA, uses the amplification of the coagulation cascade to produce a colorimetrically detectable end product. A DNA template is labelled with biotin and a haptene, and is then immobilised in a streptavidin coated microtiter plate. An antibody against the haptene is conjugated to a snake venom coagulation activator (RVVXa), and introduced into the microtiter well. The assay is then developed by the addition of coagulation factors, including an enzyme labelled fibrinogen. The deposition of enzyme labelled fibrin is indicative of a positive reaction. In this proposal we have chosen three specific applications to prove the utility of this method. They are detection of the deltaF508 mutation of cystic fibrosis, a codon 12 mutation in the first exon of K- ras for colon cancer, and the mecA gene in methicillin resistant Staphlococcus aureus.

特异性DNA序列的检测在遗传病诊断中的应用疾病、癌症和传染病正变得越来越多广泛传播。采用了一些创新技术，包括寡核苷酸连接分析和反向斑点印迹杂交方法终点，以及在一定程度上有效性这些检测的灵敏度取决于DNA检测的灵敏度使用的技术。已通过下列措施大大加强了进展：使用聚合酶链反应（PCR）。然而，执行聚合酶链式反应可能是昂贵的，以及设备和专业知识密集型。我们已应用了一种在以下领域已证明有效的技术： DNA非同位素检测的免疫测定;初始数据表示灵敏度增加10（2）-10（3）倍，常规技术。我们称之为EDNA-ELCA的检测方法使用放大凝血级联反应以产生可比色检测的最终产物。 DNA模板标记有生物素和半抗原，然后固定在包被有链霉亲和素的微量滴定板。将针对半抗原的抗体缀合至半抗原结合物。蛇毒凝血激活剂（RVVXa），并引入到微量滴定孔。然后通过添加以下物质来开发测定：凝血因子，包括酶标记的纤维蛋白原。的酶标记纤维蛋白的沉积指示阳性反应在本提案中，我们选择了三种具体应用来证明这种方法的实用性。它们是deltaF 508的检测结果囊性纤维化的突变，在第一个外显子的K-12密码子突变， ras基因用于结肠癌，mecA基因用于耐甲氧西林的结肠癌。金黄色葡萄球菌。