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 Cy5 by adjacent hybridization probes. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy number. The 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. The 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 ultraviolet illumination, red f luorescence increases after amplification in microcentrifuge 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 fluorescein/rhodamine fluorescence 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 after temperature cycling is complete, once per cycle as a monitor of product accumulation or continuously within each cycle. Sequence-specific methods (7-9) have been limited in the past to endpoint analysis. The 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 (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 intercalater YO-PRO-1 has been reported (6). To date, continuous monitoring of fluorescence within each cycle has found little use. Higuchi et al. continuously monitored amplification during 4-min temperature 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 determined. 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 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.
Three different fluorescence techniques for following rapid cycle DNA amplification are studied here using instrumentation 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 quantitative tool. Continuous monitoring within the temperature cycles of DNA amplification can reveal the mechanism of probe fluorescence, rapidly optimize tempera ture/time conditions and potentially even control temperature cycling parameters. The melting and annealing of products and probes can be followed during amplification.
Materials and methods
DNA amplification was performed in 50 mM Tris-HCl, pH 8.5 (25°C), 3 mM MgCl2, 500 μg/mL bovine serum albumin, 0.5 μM of each primer, 0.2 mM of each deoxyribonucleoside triphosphate and 0.2 U of TaqDNA polymerase per 5-μL sample, unless otherwise stated. Human genomic DNA (denatured for 1 min by boiling) or purified amplification product was used as DNA template. Purified amplification product was obtained by phenol /chloroform extraction and ethanol precipitation (17), followed by removal of primers by repeated washing through a Centricon 30 microconcentrator (Amicon, Beverly, MA, USA). Template concentrations were determined by absorbance at 260 nm. A260/A280 ratios of templates were greater than 1.7.
Primers were synthesized by standard phosphoramidite chemistry (Gene Assembler Plus; Pharmacia Biotech, Piscataway, NJ, USA). SYBR Green I was obtained from Molecular Probes (Eugene, OR, USA). The β-actin primers and fluorescein/rhodamine dual probe were obtained from Perkin- Elmer (Norwalk, CT, USA). The human β-globin primers RS42/KM29 (536 bp) and PC03/PC04 (110 bp) have been described previously (19). The single-labeled probes 5′-CAAACAG ACACCATGGTGCACCTGACTCCTGAGGA- fluorescein-3′and 5′-Cy5- AAGTCTGCCGTTACTGCCCTGTGGGGCAAG- phosphate-3′ were synthesized using a fluorescein phosphoramidite (Glen Research, Sterling, VA, USA), a Cy5 phosphoramidite (Pharmacia Biotech) and a chemical phosphorylation reagent (Glen Research). These adjacent probes hybridize internal to the PC03/ PC04 β-globin primer pair on the same DNA strand and are separated by one base pair. Probes were purified by reverse-phase C-18 high pressure liquid chromatography, and homogeneity was checked by polyacrylamide gel electrophoresis and absorbance (A260 and the absorbance maximum of the fluorophore). The β-actin hydrolysis probe and the β-globin hybridization probes were used at 0.2 μM each. Figure 1 schematically compares the differences between the three fluorescence monitoring techniques: dsDNA-specific dyes, hydrolysis probes and hybridization probes.