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fluorescein and Cy5 are available for direct incorporation during automated synthesis. Signal generation requires two independent hybridization events to occur each cycle and is more directly related to product concentration than cumulative hydrolysis probes. However, aſter many cycles, the fluorescence from hybridization probes decreases (Figure 4), possibly because of probe consumption by exonuclease hydrolysis. We are not aware of prior reports using


fluorescein and Cy5 as a resonance energy transfer pair, although phycoerythrin and Cy7 (Amersham International, Little Chalfont, Bucks, England, UK) (with similar spectral separation) have been used as a bright tandem dye in immunofluorescence (13). With fluorescein and Cy5, the spectral overlap is small, but the molar absorption coefficient and absorption wavelengths of Cy5 are high. All three factors (overlap, absorptivity and wavelength) contribute to the overlap integral that determines energy transfer rates (24). Cy5 also has low absorbance at fluorescein excitation wavelengths (11), reducing direct excitation of the acceptor. Many aspects of hydrolysis and


hybridization probes remain to be studied. The effects of probe length, melting temperature and concentration, distance between hybridization probes, distance to primers, temperature profiles, acquisition point within a cycle and type of polymerase have not been systematically optimized. DNA amplification is extensively


used but not rigorously understood. Continuous f luorescence monitoring provides an instantaneous window into the amplification process. For example, product denaturation occurs in less than 1 s (20,22), yet most protocols call for 10 s to 1 min of denaturation. By monitoring with dsDNA- specific dyes, product denaturation can be observed during each amplification cycle (Figure 5), a convincing demonstration that most denaturation protocols are excessive. To give another example, many causes of the “plateau effect” have been proposed, but little data are available to distinguish between alternatives. Figure 5 shows that product-to-product annealing is very rapid. In fact, during later cycles of amplification, a majority of product anneals to itself each cycle during cooling before the primer annealing temperature has been reached. Tis rapid reannealing is observed with cooling rates of 5°–10°C/s, characteristic of rapid cycling. Product reannealing with slower, conventional temperature cyclers


Vol. 54 | No. 6 | 2013 319 www.BioTechniques.com


would be greater. Product-to-product annealing appears to be a major, and perhaps the sole, cause of the “plateau effect”. As previously suggested (22), continuous


fluorescence monitoring within each temperature cycle can be used to control temperature cycling parameters. With dsDNA-specific dyes, amplification can be stopped after a certain amount of product is synthesized, thus avoiding overamplification of alternative templates. Te extension phase of each cycle needs to


be continued only as long as fluorescence increases. Product denaturation can be assured each cycle by increasing the temperature until the fluorescence reaches baseline. Tis kind of fluorescence feedback should allow very rapid optimization of new assays. Limiting the time that product is exposed to denaturation temperatures may also be useful for the amplification of long products (1,2). Additional uses of continuous monitoring


with fluorescent dyes can be envisioned. For example, with fine temperature control


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