Nanoliter scale real-time PCR/CE for cancer detection

项目描述（申请人提供）：我们拟开发一套高通量实时PCR/毛细管电泳（PCR/CE）系统，用于高灵敏度、定量检测癌症分子标志物，并应用该系统对前列腺癌患者空尿中单个癌细胞端粒酶活性和基因表达进行定量分析。在提出的系统中，我们将使用一次性单块多毛细管阵列（MMCA）作为实时PCR的容器。MMCA的使用将使高灵敏度的低体积（经济）PCR/CE分析以及基于终端稀释和泊松统计分析的临床样品中DNA目标的绝对定量成为可能。该系统的超高灵敏度将是由于使用单光子检测荧光标记的DNA物种。系统操作将实现自动化，并将依靠现代数据采集、处理和处理技术（互联网可访问性、标准数据库方法等）。我们将对所提出的系统进行临床评估，用于无创前列腺癌早期检测，并确定端粒酶活性定量分析用于区分良性和恶性增生的可行性。

DESCRIPTION (provided by applicant): We propose to develop an integrated high throughput real-time PCR/capillary electrophoresis (PCR/CE) system for highly sensitive, quantitative detection of molecular markers of cancer, and apply this system for quantitative analysis of the telomerase activity and gene expression in single cancer cells isolated from voided urine of prostate cancer patients. In the proposed system, we will use disposable monolith multi-capillary arrays (MMCA) as a vessel for realtime PCR. The use of MMCA will enable highly sensitive low volume (economical) PCR/CE assays as well as absolute quantification of DNA target in clinical samples based on analysis of terminal dilutions and Poisson statistics. Ultra high sensitivity of the system will be due to the use of single photon detection of fluorescently labeled DNA species. The system operation will be automated and will rely on modern data acquisition, processing, and handling technologies (internet accessibility, standard database approach, etc.) We will carry out the clinical evaluation of the proposed system for non-invasive early detection of prostate cancer, and determine the feasibility of quantitative analysis of telomerase activity for discrimination between benign and malignant hyperproliferation.