INTEGRATED FIBER OPTIC SENSOR FOR DNA HYBRIDIZATION

该项目第一阶段进行的研究将证明在带有荧光检测的光纤传感器(FOS)上进行动态DNA杂交分析的可行性。FOS安装在温度控制的反应池中。DNA序列将使用聚合酶链式反应(PCR)进行扩增，并与固定在光纤传感器上的特定探针杂交。杂交事件将通过测量用于聚合酶链式反应的标记核酸(DNTPs)的荧光发射来监测。第二阶段将包含与靶序列一起共扩增的内标(IS)序列。IS与同一FOS上的不同探测器杂交。使用荧光能量转移和双色荧光检测器，可以使扩增事件标准化。原型仪器将是独立的，只需要将样品施加到包含FOS和消耗性生化试剂的反应池中。该系统将允许对病毒和其他感染源进行定量聚合酶链式反应，确定转移情况，进行突变分析和其他基因测定。FOS将可重复使用并易于校准，作为模型系统开发的探头将允许对制造进行质量控制。拟议的商业应用：这一集成的核酸扩增和检测系统提供了现有方法所没有的功能，包括原位扩增、以均相分析格式实时监测杂交、通过内标进行定量、探针重复使用、易于质量控制和仪器便携性。该系统可以适应多种扩增形式，并有可能用于遗传学研究实验室和医院实验室的临床检测。

The research performed in Phase I of this project will demonstrate the feasibility of performing a kinetic DNA hybridization assay on a fiber optic sensor (FOS) with fluorescence detection. The FOS is mounted within a temperature controlled reaction cell. DNA sequences will be amplified using the polymerase chain reaction (PCR) and hybridized to specific probes immobilized to fiber optic sensors. The hybridization event will be monitored by measuring the fluorescence emission of labeled nucleic acids (dNTPs) that are used in PCR. Phase II will incorporate an internal standard (IS) sequence which is coamplified along with the target sequence. The IS hybridizes to a different probe on the same FOS. Using fluorescence energy transfer and a two-color fluorescence detector, IS allows normalization of the amplification event. The prototype instrument will be self-contained, requiring only the application of sample to a reaction cell that contains the FOS and the expendable biochemical reagents. This system will allow quantitative PCR for viruses and other infectious agents, determination of metastasis, mutational analysis and other genetic determinations. The FOS will be reusable and readily calibrated, and probes that are developed as a model system will allow for quality control of manufacturing. PROPOSED COMMERCIAL APPLICATIONS: This integrated nucleic acid amplification and detection system provides features not found in available methods, including in situ amplification, real-time monitoring of hybridization in a homogeneous assay format, quantification by means of an internal standard, re-use of probes, ease of quality control, and instrument portability. The system can be adapted to a variety of amplification formats, and has the potential for use in genetics research laboratories and for clinical determinations in hospital labs.