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plot is shown for amplification with the β-actin exonuclease probe. During each cycle, the fluorescence ratio varies linearly with temperature and there is little, if any, hysteresis. Although the fluorescence of both fluorescein and rhodamine decreases with increasing temperature (data not shown), the rate of change is greater for rhodamine, resulting in an increasing ratio with increasing temperature. Te fluores- cence ratio increases during the annealing/ extension phase at a constant temperature when probe hydrolysis is presumed to


occur. In contrast, with adjacent hybrid- ization probes, the fluorescence signal is dependent only on hybridization, not hydrolysis. Fluorescence vs. temperature plots of amplification with hybridization probes show obvious hysteresis (Figure 7). During heating to product denaturation temperatures, the probes appear to disso- ciate between 65° and 75°C, returning the fluorescence ratio to background levels. These temperature-dependent hybrid- ization effects are not apparent with the 5´-exonuclease probe (Figure 6).


Discussion


Tree different chemistries for fluorescent monitoring of DNA amplification have been studied: a dsDNA-specific dye, a dual-labeled exonuclease hydrolysis probe and adjacent hybridization probes. Te hydrolysis and hybridization probes are sequence specific. Hydrolysis probes require one hybridization event for signal generation, whereas hybridization probes require two independent hybridizations. Figure 2 schematically compares and contrasts the three fluorescence monitoring systems. dsDNA-specific dyes such as ethidium bromide (3,4,22) or SYBR Green I can be used as generic indicators of amplification. We used SYBR Green I instead of ethidium bromide because it has an excitation maximum near fluorescein and, in our hands, gave a stronger signal with DNA than excitation of ethidium bromide with visible light. These dyes depend on the specificity inherent in the amplification primers. Currently, nonspecific amplification after many cycles limits detection sensitivity to about 100 initial template copies (Figure 2, see also Reference 4). Improvements in amplification specificity could remove this limitation. When low-copy-number detection


and quantification are needed, additional specificity can be provided by sequence- specific fluorescent probes. Hydrolysis of a dual-labeled exonuclease probe is sequence specific (8,9). However, the design, synthesis and purification of dual-labeled hydrolysis probes require care. Hybridization is a necessary but not sufficient condition for hydrolysis; all probes are not cleaved efficiently. Synthesis of the dual-labeled probes involves manual addition of the rhodamine label, and at least one stage of high-pressure liquid chromatography is required for purification. In addition, the signal generated by exonuclease probes is cumulative and only indirectly related to product concentration. Hence, the fluorescence signal continues to increase even after the amount of product has reached a plateau (Figures 3 and 6). Instead of depending on hydrolysis of a dual-labeled probe, hybridization can be detected directly through resonance energy transfer as outlined by Morrison (10). By using two adjacent probes labeled separately with fluorescein and Cy5, energy transfer to Cy5 increases with product accumulation (Figures 4 and 7). In contrast to exonuclease probes, probe synthesis is relatively simple because amidites for both


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