INTEGRATED FIBER OPTIC SENSOR FOR DNA HYBRIDIZATION

在该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杂交分析的可行性。FOS安装在温度控制的反应池内。DNA序列将使用聚合酶链反应（PCR）扩增，并杂交到固定在光纤传感器上的特定探针上。杂交事件将通过测量用于PCR的标记核酸（dNTPs）的荧光发射来监测。二期将纳入一个内部标准（IS）序列，该序列与目标序列一起被共扩增。IS与同一FOS上的不同探针杂交。利用荧光能量转移和双色荧光检测器，IS允许放大事件的规范化。原型仪器将是独立的，只需要将样品应用到包含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因子进行定量PCR，确定转移，突变分析和其他遗传确定。FOS将可重复使用并易于校准，并且作为模型系统开发的探针将允许制造质量控制。拟议的商业应用：这种集成的核酸扩增和检测系统提供了现有方法所没有的功能，包括原位扩增，均匀分析格式的杂交实时监测，通过内部标准进行定量，探针的重复使用，易于质量控制和仪器便携性。该系统可以适应多种扩增格式，并有潜力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.