Multiwell colorimetric assay for DNA repair protein.

描述(申请人提供)：DNA损伤是致癌过程中的关键事件。在肿瘤发生和/或癌症治疗过程中，修复受损DNA的缺陷意味着预后不佳。除了监测DNA损伤的形成外，它还有助于监测DNA修复蛋白的活性。我们建议开发一种新技术，允许高通量和快速分析最常见的DNA修复蛋白。我们将使用固定在96孔微孔板上的受损DNA探针来捕获细胞或组织提取物中的DNA修复蛋白。将合成六种含有不同DNA损伤类型的生物素化寡核苷酸。利用纯化的重组DNA修复蛋白建立DNA与蛋白质结合的条件。针对DNA修复蛋白产生的抗体将在两小时内识别结合蛋白并产生定量结果。最后，该程序将应用于相关样本，如损伤剂处理的细胞系和疾病模型的提取物。这种分析将有助于阐明DNA损伤和修复途径的分子机制。它还将为科学家和临床医生提供一种独特的工具，在通常所需时间的一小部分内回答风险评估中的开创性问题。

DESCRIPTION (provided by applicant): DNA damages are key events during carcinogenesis. Deficiencies in repairing damaged DNA represent a poor prognosis during tumorigenesis and/or cancer treatment. Besides monitoring DNA damage formation, it would be helpful to monitor DNA repair protein activity. We propose to develop a new technology that allows for high-throughput and fast analysis of the most common DNA repair proteins. We will use a damaged DNA probe immobilized onto a 96-well microplate to capture DNA repair proteins in an extract from cells or tissues. Six biotinylated oligonucleotides containing different DNA damage types will be synthesized. The DNA-protein binding conditions will be developed by using purified recombinant DNA repair proteins. Antibodies raised specifically against DNA repair proteins will identify the bound protein and yield a quantitative result in two hours. Finally, the procedure will be applied to relevant samples such as extracts from damaging agent-treated cell lines and disease models. Such assay will help elucidate the molecular mechanisms of the DNA damage and repair pathways. It would also provide scientists and clinicians with a unique tool to answer seminal questions in risk assessment in a fraction of the time normally required.