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and dsDNA-specific dyes, product purity could be estimated by melting curves. With rapid temperature control, absolute product concentration could be determined by product- to-product annealing kinetics. The only requirements are fluorescence monitoring, the ability to change temperatures rapidly and strict intra-sample temperature homogeneity. Aspects of instrument design are discussed elsewhere (23). Conventional end-point analysis of DNA

amplification by gel electrophoresis identifies product size and estimates purity. However, because amplification is at first stochastic, then exponential, and finally stagnant, the utility of end-point analysis is limited for quantifi- cation. Fluorescence monitoring every cycle during DNA amplification is an extraordi- narily powerful technique for quantification. With simple instrumentation and fluorescent monitoring each cycle, sequence-specific detection and quantification can be achieved in 5–20 min aſter temperature cycling has begun. Although the final fluorescence signal is decreased when low copy numbers are amplified, quantification between 0 and 1000 initial template copies appears possible (Figures 3 and 4). Tese techniques should be particularly useful in assays where rapid quanti- fication is desired, such as in the amplification of clinical serum viruses.


Tis work was financially supported by an STTR grant from the NIH (1 R41 GM51647), a Technology Innovation grant from the University of Utah Research Foundation, a Biomedical Engineering grant from the Whitaker Foundation, Idaho Technology, and Associated Regional and University Pathologists. We thank Marla Lay, Gundi Reed, Douglas Searles and Charles Hussey for technical assistance and insightful conversation.


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