Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification

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Abstract
Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR® Green I, (ii) a decrease in fluorescein quenching by rhodamine after exonuclease cleavage of a dual-labeled hydrolysis probe and (iii) resonance energy transfer of fluorescein to Cy5TM by adjacent hybridization probes. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy number. The sensitivity of SYBRGreen I detection is limited by nonspecific product formation. Use of a single exonuclease hydrolysis probe or two adjacent hybridization probes offers increasing levels of specificity. In contrast to fluorescence measurement once per cycle, continuous monitoring throughout each cycle monitors the temperature dependence of fluorescence. The cumulative, irreversible signal of hydrolysis probes can be distinguished easily from the temperature-dependent, reversible signal of hybridization p...

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