CONTINUOUS FLUORESCENCE MONITORING OF RAPID CYCLE DNA AMPLIFICATION

Rapid cycle DNA amplification was continuously monitored by three diff erent fluorescence techniques. Fluorescence was monitored by (i) the d ouble-strand-specific dye SYBR(R) Green I, (ii) a decrease in fluoresc ein quenching by rhodamine after exonuclease cleavage of a dual-labele d hydrolysis probe and (iii) resonance energy transfer of fluorescein to Cy5(TM) by adjacent hybridization probes. Fluorescence data acquire d once per cycle provides rapid absolute quantification of initial tem plate copy number. The sensitivity of SYBR Green I detection is limite d by nonspecific product formation. Use of a single exonuclease hydrol ysis probe or two adjacent hybridization probes offers increasing leve ls of specificity. In contrast to fluorescence measurement once per cy cle, continuous monitoring throughout each cycle monitors the temperat ure dependence of fluorescence. The cumulative, irreversible signal of hydrolysis probes can be distinguished easily from the temperature-de pendent, reversible signal of hybridization probes. By using SYBR Gree n I, product denaturation, annealing and extension can be followed wit hin each cycle. Substantial product-to-product annealing occurs during later amplification cycles, suggesting that product annealing is a ma jor cause of the plateau effect. Continuous within-cycle monitoring al lows rapid optimization of amplification conditions and should be part icularly useful in developing new, standardized clinical assays.