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.
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.
| Nuovo, G J; Hohman, R J; Nardone, G A et al. (1999) In situ amplification using universal energy transfer-labeled primers. J Histochem Cytochem 47:273-80 |
