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fluorescein fluorescence ratio increases. Te change in fluorescence ratio during hybridization is largely due to an increase in Cy5 fluorescence from resonance energy transfer (data not shown). In contrast to hydrolysis probes, the fluorescence signal of hybridization probes tends to decrease with excessive cycling. No fluorescence signal is generated in the absence of template. Sequence-specific probes have an even
greater dynamic range for template quanti- fication than do dsDNA dyes. As the template copy number decreases below 103
,
signal intensity decreases because specific amplification efficiency decreases, but low copy numbers can still be quantified because the negative control signal is stable (Figures 3 and 4). Although multiple samples would need to be run to confirm Poisson statistics, it appears that these specific techniques can discriminate a single initial template copy from negative controls (compare 0 and 1 average initial template copies in Figures 3 and 4). With each technique, the fluorescence
response is not strictly proportional to the amount of specific product. With SYBR Green I, two factors nonspecific amplifi- cation of alternative templates results in fluorescence unrelated to specific product, particularly aſter many cycles (Figure 2). In addition, when the fluorescence of purified DNA standards is measured, the response is only linear to 10–20 ng of DNA per 5-µL reaction under the conditions used (data not shown). Higher concentrations of DNA show proportionally less fluores- cence, presumably because the amount of SYBR Green I becomes limiting. Higher concentrations of SYBR Green I than those used here (<1:7000 dilution) inhibit ampli- fication (data not shown). With hydrolysis probes, the fluorescence signal continues to increase after the plateau phase has been reached (Figure 3). With hybrid- ization probes, the fluorescence decreases during the plateau phase (Figure 4). Despite the nonlinearity of these f luorescence techniques, they are very useful for absolute quantification of initial template copy number when fluorescence is measured for each amplification cycle (Figures 2–4). Because fluorescence depends on temper-
ature, fluorescence is usually acquired only once per cycle at a constant temperature to monitor product yield. Tis eliminates any confounding effect of temperature on fluorescence. However, interesting and useful information about hybridization can be obtained by monitoring fluorescence continuously throughout each temperature cycle. For example, fluorescence vs. temper- ature plots of amplification with SYBR Green I show the temperature dependence
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of strand status during DNA amplification (Figure 5). Early cycles appear identical, with a nonlinear increase in fluorescence at lower temperatures. As amplification proceeds, later cycles appear as rising loops between annealing and denatur- ation temperatures that show significant hysteresis. By hysteresis, we mean that the observed fluorescence during heating is greater than that during cooling. As the sample is heated, fluorescence is high until denaturation occurs (apparent as a sharp
drop in fluorescence). As the sample cools from denaturation to annealing temper- atures, f luorescence increases rapidly, apparently reflecting product-to-product annealing. Fluorescence also increases during extension while the temperature is held constant. Fluorescence vs. temperature plots
of 5´-exonuclease probes confirm that probe hydrolysis (not hybridization) is the mechanism of signal generation. In Figure 6, a fluorescence vs. temperature
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