This is a proposal to develop a fluorescence based method for detection of amplification products in a closed system. This method is based on the incorporation of energy transfer (ET)-labeled primers into the amplified DNA. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into the amplification product. This technology eliminates carry-over contamination, simplifies the amplification assay, and opens up new possibilities for cost-effective automated devices for clinical practice. The Phase I research has demonstrated the feasibility and applications of this technology (Sunrise, Oncor). The objectives of the Phase II project include: i) development of a universal detection system using a single ET-labeled primer for several targets; ii) a multiplex assay with hairpin primers of different colors; iii) an automated scheme for chemical synthesis of ET-labeled primers to simplify production; iv) application of the amplification detection system in real-time for target quantification; and v) diagnostic applications of ET-labeled hairpin primers that include telomerase detection, quantification of DNA or RNA targets, RT PCR, in situ PCR, allele-specific PCR, and detection of methylated DNA. A long term objective is a fully automated robotics amplification detection system.
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