Reports BioTechniques 30th Anniversary GEM
Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification
Carl T. Wittwer, Mark G. Herrmann, Alan A. Moss and Randy P. Rasmussen University of Utah, Salt Lake City, UT, USA Reprinted from: BioTechniques 22:130-138 (January 1997)
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 aſter exonuclease cleavage of a dual-labeled hydrolysis probe and (iii) resonance energy transfer of fluorescein to Cy5 by adjacent hybridization probes. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy number. Te sensitivity of SYBR Green 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. Te cumulative, irreversible signal of hydrolysis probes can be distinguished easily from the temperature-dependent, reversible signal of hybridization probes. By using SYBR Green I, product denaturation, annealing and extension can be followed within each cycle. Substantial product-to-product annealing occurs during later amplification cycles, suggesting that product annealing is a major cause of the plateau effect. Continuous within-cycle monitoring allows rapid optimization of amplification conditions and should be particularly useful in developing new, standardized clinical assays.
Fluorescent probes can be used to detect and monitor in vitro DNA amplification (14). Useful probes include double- stranded DNA (dsDNA)-specific dyes and sequence-specific probes. With the intercalater ethidium bromide and ultra- violet illumination, red f luorescence increases aſter amplification in micro- centrifuge tubes (3) or capillaries (22). Sequence-specific fluorescence detection is possible with oligonucleotide probes. For example, dual-labeled fluorescein/ rhodamine probes may be cleaved during polymerase extension by 5´-exonuclease activity, separating the fluorophores and increasing the f luorescein/rhodamine f luorescence ratio (5,8,9). Alternately, “molecular beacons” have been described with a fluorogenic conformational change when hybridized to their targets (7). Fluorescence can be measured aſter
temperature cycling is complete, once per cycle as a monitor of product accumu- lation or continuously within each cycle. Sequence-specific methods (7–9) have been limited in the past to endpoint analysis. Te potential of once per cycle monitoring for quantification of initial template copy number was first suggested and developed by Higuchi et al. using ethidium bromide
Vol. 54 | No. 6 | 2013
(3,4). Fluorescence is acquired during the extension or combined annealing/ extension phase of each cycle and is related to product concentration. A quantitative assay for hepatitis C RNA using the inter- calater YO-PRO-1 has been reported (6). To date, continuous monitoring of fluores- cence within each cycle has found little use. Higuchi et al. continuously monitored amplification during 4-min temper- ature cycles using a 10-s integration time (3). An inverse correlation of ethidium fluorescence to temperature was noted, with product accumulation resulting in increased fluorescence during annealing/ extension. If f luorescence is continuously
monitored within each temperature cycle, the hybridization of amplification products and probes can be followed during amplification. With dsDNA dyes, product denaturation and reannealing can be monitored. With probes that change fluorescence upon hybridization, probe melting temperatures can be deter- mined. With rapid, homogeneous control of sample temperature, the kinetics of hybridization can be followed. We have previously used capillaries and forced-air heating for precise temperature control
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that allows 30 cycles in less than 15 min (18–22). By minimizing denaturation and annealing times, the specificity and yield of such “rapid cycle” amplifications are also improved (2,12,15,16,20–22). In addition to facilitating rapid heat transfer, glass capillaries are optically clear and make natural cuvettes for fluorescence analysis. Tree different fluorescence techniques
for following rapid cycle DNA amplifi- cation are studied here using instrumen- tation described elsewhere (23). Instead of ethidium bromide, SYBR Green I is used as a dsDNA specific dye. A 5´-exonuclease probe is then compared to SYBR Green I monitoring. Finally, a novel fluorescence scheme based on adjacent hybridization probes with resonance energy transfer from fluorescein to the cyanine dye Cy5 (11) is demonstrated. Once per- cycle monitoring of multiple samples is a powerful quanti- tative tool. Continuous monitoring within the temperature cycles of DNA amplifi- cation can reveal the mechanism of probe fluorescence, rapidly optimize tempera ture/time conditions and potentially even control temperature cycling parameters. Te melting and annealing of products and probes can be followed during ampli- fication.
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