ON-CHIP DNA PURIFICATION/PCR FOR CANCER DIAGNOSTICS

描述：（申请人描述）分子生物学进展 技术为许多疾病的诊断提供了分子基础， 并催生了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）扩增DNA和电泳 用于检测扩增产物的分离，是劳动密集型的， 耗时且不适于自动化。 这种方法论 “瓶颈”可以通过毛细管电泳（CE）部分解决， 更全面地通过“芯片电泳”。 芯片电泳 涉及在蚀刻在一个微通道中的微米级通道中进行CE分离， 平面玻璃表面。 此外，Co允许快速分离， 由于小型化，“芯片”还提供了一个方便的平台， 用于整合电泳前样品处理。 因此，样品 制备、PCR扩增和电泳分离/检测， 合并成一个单一的微量分析装置。 的一部分 为了开发用于癌症诊断的集成电泳芯片，我们 计划开发一种适合芯片的热循环方法。 使用钨 灯作为红外热源，将实现非接触式温度循环 在芯片的聚焦区域执行，并完成温度控制 通过遥感。 这将涉及发展一个IR 感测装置（高温计）或折射率检测方案，两者均 这将提供骑自行车的远程控制。 因为这很重要 自动化从样品中提取DNA用于芯片PCR，我们还计划 开发一种适合芯片的方法，用于从血液中纯化DNA， PCR和电泳。 该提案的重点将是明确 固相萃取法纯化白色DNA 血细胞的方式，可以很容易地推断到芯片。 证明 芯片上DNA纯化的可行性。 扩增和检测 癌症特异性DNA标记将使用急性骨髓性 白血病和B细胞淋巴瘤作为模型系统。

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.