ON-CHIP DNA PURIFICATION/PCR FOR CANCER DIAGNOSTICS

用于癌症诊断的芯片 DNA 纯化/PCR

• 批准号: 2896660
• 负责人: STEPHEN G. WEBER
• 金额: $ 5.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2896660
• 关键词: B cell lymphoma; DNA; acute myelogenous leukemia; biomedical equipment development; capillary electrophoresis; chromosome translocation; diagnosis design /evaluation; gene rearrangement; genetic markers; human genetic material tag; human tissue; immunoglobulin genes; leukocytes; neoplasm /cancer diagnosis; neoplasm /cancer genetics; nucleic acid sequence; polymerase chain reaction

DESCRIPTION: (Applicant's Description) Advances in molecular biological techniques have provided a molecular basis for the diagnosis of many diseases and spawned the era of molecular diagnostics for cancer diagnosis and prognosis. The main methods associated with molecular diagnostics, polymerase chain reaction (PCR) for amplification of DNA and electrophoretic separation for detection of the amplified products, are labor-intensive, time- consuming and not amenable to automation. This methodological "bottleneck" can be solved, in part, by capillary electrophoresis (CE) and more comprehensively by "chip electrophoresis". Chip electrophoresis involves carrying out CE separations in micron-scale channels etched in a planar glass surface. In addition Co allowing for rapid separations as a result of miniaturization, the "chips" also provide a convenient platform for integrating pre-electrophoresis sample processing. Consequently, sample preparation, PCR amplification and electrophoretic separation/detection can be amalgamated into a single microanalysis device. As part of the development of an integrated electrophoretic chip for cancer diagnostics, we plan to develop a chip-amenable approach to thermocycling. Using a tungsten lamp as an infrared heat source, noncontact temperature cycling will be executed on focused areas of the chip with temperature control accomplished through remote sensing. This will involve either the development of an IR sensing device (pyrometer) or a refractive index detection scheme, both of which will provide remote control of cycling. Since it will be important to automate the extraction of DNA from samples for on-chip PCR, we also plan to develop a chip-amenable methodology for purifying DNA from blood for on-chip PCR and electrophoresis. The focus of this proposal will be to explicit solid phase extraction methodologies for purification of DNA from white blood cells in a manner that can easily be extrapolated to the chip. Proof of feasibility for on-chip DNA purification. amplification and detection of cancer-specific DNA markers will be accomplished using acute myelogenous leukemia and B-cell lymphoma as model systems.

描述：（申请人的描述）分子生物学的进步 技术为诊断许多人提供了分子基础 疾病并产生了用于癌症诊断的分子诊断时代 和预后。 与分子诊断相关的主要方法， 聚合酶链反应（PCR）进行DNA和电泳的扩增 用于检测放大产品的分离是劳动密集型的， 耗时，不适合自动化。 这个方法论 毛细管电泳（CE）和 更全面地通过“芯片电泳”。 芯片电泳 涉及在蚀刻在A中的微米级通道中进行CE分离 平面玻璃表面。 另外，可以将快速分开作为 小型化的结果，“芯片”还提供了方便的平台 用于整合电子样品前样品处理。 因此，样品 准备，PCR扩增和电泳分离/检测可以 将其合并到单个微分析装置中。 作为一部分 开发用于癌症诊断的综合电泳芯片，我们 计划开发一种芯片不明的方法来进行热环。 使用钨 灯作为红外热源，非接触式温度循环将是 在芯片的聚焦区域执行，并完成了温度控制 通过遥感。 这将涉及IR的开发 传感设备（高温计）或折射率检测方案，这两个方案 这将提供骑自行车的遥控器。 因为很重要 自动从样品中提取DNA的芯片PCR，我们还计划 开发一种可从片上纯化DNA的芯片不明的方法 PCR和电​​泳。 该提案的重点是明确 固体提取方法用于纯化白色DNA 血细胞以很容易被外推到芯片的方式。 证明 片上DNA纯化的可行性。 放大和检测 癌症特异性的DNA标记将使用急性骨髓 白血病和B细胞淋巴瘤作为模型系统。