SBIR-STTR Award

Although nucleic acid amplification is an important diagnostic tool, its use in clinical practice is limited by the complexity of protocols and the problem of carry-over contamination, which is a source of false- positive results. We suggest a new method for direct detection of amplification products in a closed system. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The primers are designed in such a way that the fluorescent signal is generated only when the primers are incorporated into the amplification product. The proposed technology can eliminate carry-over contamination, simplify the amplification assay, and open up new possibilities for cost-effective automated devices for amplification. The feasibility of this technique has been demonstrated in preliminary experiments. The objective of this Phase I project include: i) determination of optimal structures of oligonucleotide primers and locations of energy transfer labels giving highest signal to background ration along with the maximum amplification efficiency; ii) determination of the laboratory equipment for the simultaneous and quantitive detection for the large amount of reactions. iii) comparison of the sensitivity of the proposed method with traditional amplification/detection techniques. In Phase II study we will assess the clinical utility of this method. PROPOSED COMMERCIAL APPLICATIONS: Detection of amplification product in a "closted tube format" can be applied to any diagnostic procedure where a "yes or no" answer is required. For example, detection of infectious disease agents, some forms of cancer or contamination of food or water with a specific microorganisms. The proposed method will be less costly than presently used amplification kis, will require less skill form the technician, and will decrease the chance of false-positive results. A fully automated diagnostic systems can be created based on the proposed methods.

Thesaurus Terms:
fluorescent dye /probe, gene amplification, method development, polymerase chain reaction DNA primer, complementary DNA, nucleic acid structure, oligonucleotide fluorescence spectrometry, gel electrophoresis, nucleic acid chemical synthesisNational Institute of General Medical Sciences (NIGMS)

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.

Thesaurus Terms:
biomedical automation, fluorescent dye /probe, gene amplification, method development, polymerase chain reaction DNA primer, complementary DNA, nucleic acid structure, oligonucleotide nucleic acid chemical synthesis