INTEGRATED FIBER OPTIC SENSOR FOR DNA HYBRIDIZATION

在本项目的第一阶段进行的研究将证明在具有荧光检测的光纤传感器（FOS）上进行动态DNA杂交测定的可行性。FOS安装在温度受控的反应单元内。DNA序列将使用聚合酶链式反应（PCR）扩增，并与固定在光纤传感器上的特异性探针杂交。 通过测量PCR中使用的标记核酸（dNTP）的荧光发射来监测杂交事件。第II阶段将加入内标（IS）序列，该序列与靶序列一起沿着共扩增。IS与同一FOS上的不同探针杂交。使用荧光能量转移和双色荧光检测器，IS允许扩增事件的归一化。原型仪器将是独立的，只需要将样品应用于包含FOS和消耗性生化试剂的反应池。该系统将允许对病毒和其他感染因子进行定量PCR，确定转移，突变分析和其他遗传测定。FOS将可重复使用并易于校准，并且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：这种集成的核酸扩增和检测系统提供了在现有方法中没有发现的特征，包括原位扩增、以均相测定形式实时监测杂交、通过内标进行定量、探针的重复使用、易于质量控制和仪器便携性。该系统可以适应各种扩增形式，并有可能用于遗传学研究实验室和医院实验室的临床测定。

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.