Fluorescent dyes for use with the

Detection of Multiple Reporter Dyes in Real-time, On-line PCR Analysis with the LightCycler System Gregor Sagner, Cornelia Goldstein, and Rob van Miltenburg Roche Molecular Biochemicals, Penzberg, Germany Introduction The LightCycler System is a fast PCR amplification and analysis system. Central to the function of the LightCycler System are the use of air as a medium for heating and cooling, and the use of capillaries as reaction vessels for efficient temperature exchange between the air and the reagents contained in the capillary. The capillaries are made of borosilicate glass and can therefore also be used as a cuvette. Addition of selected fluorescent dyes to the reaction components allows the PCR to be monitored in real-time and on-line. Fluorescence data are recorded by the optical unit of the instrument, are displayed on a computer screen as PCR proceeds, and are stored for further analysis. A detailed description of the LightCycler System and real-time on-line monitoring of PCR can be found at: http://biochem.roche.com/lightcycler The most prominent fluorescent formats used as reporters in the LightCycler System are the doublestranded DNA binding dye SYBR Green I, and the so-called Hybridization Probes format. Whereas signal development in the presence of the SYBR Green I dye is dependent on the formation of double-stranded DNA, regardless of the DNA sequence, the Hybridization Probes format requires 2 specially designed, sequencespecific oligonucleotides. These Hybridization Probes, labeled with two different fluorescent molecules, hybridize next to each other on the target DNA molecule (Figure 1). The first fluorescent dye, the donor dye fluorescein, is excited at 470 nm by the light source of the LightCycler Instrument. Instead of emitting light at 530 nm, the fluorescein can transfer its energy in a nonfluorescent manner to an acceptor dye, for example LightCycler-Red 640 (LC-Red 640) or, alternatively, LightCycler-Red 705 (LC-Red 705), in a process called Fluorescence Resonance Energy Transfer (FRET). The acceptor or reporter dye emits light of a longer wavelength and the intensity of this signal can be correlated with the amount of target DNA molecules. Efficient FRET can only take place when the 2 dyes are in direct local proximity and when the emission spectrum of the donor dye overlaps the absorption spectrum of the reporter dye. In this article, we describe the simultaneous use of two reporter dyes, allowing the measurement of two independent target sequences. Fluorescent dyes for use with the LightCycler System The LightCycler Instrument s optical unit, a microvolume fluorimeter (Figure 2), is capable of measuring fluorescence in three separate channels simultaneously. The choice of band pass filters and dichroic mirrors for the optical unit is optimized to the fluorescent dyes that are used. Channel 1 measures fluorescence (F1) at Figure 1: Hybridization Probes format. 7

Figure 2: Microvolume fluorimeter of the LightCycler Instrument 530 nm and is the channel used for SYBR Green I and for fluorescein. Channel 2 (F2; 640 nm) is used to measure signals from LC-Red 640. Channel 3 (F3; 710 nm) is designed for use with LC-Red 705. The optical unit for the LightCycler System was developed with the objective of performing dual color detection. To achieve this objective, fluorescent dyes with optimized spectral separation had to be developed. The spectra of the dyes are shown in Figure 3. The spectral separation of excitation and emission spectra of the two FRET acceptor dyes (LC-Red 640 and LC-Red 705) is optimized to simultaneously: provide minimized spectral crosstalk from each dye into the other detection channels (e.g., a minimal fluorescence signal measured in channel 3 coming from LC-Red 640), provide adequate overlap of the excitation spectrum of the respective acceptor dyes with the fluorescein emission spectrum to allow efficient FRET. In meeting both objectives, a residual crosstalk of emitted light from LC-Red 640 into channel 3 cannot be avoided. Minor crosstalks from LC-Red 705 into channel 2 and from fluorescein into channels 2 and 3 occur as well. When measuring the two dyes simultaneously, correction of this crosstalk must be performed in order to obtain signals that can directly be correlated with the amount of hybridized probes and thus with the amount of the respective target. This color compensation is achieved by correcting the measured raw fluorescence values. The LightCycler-Color Compensation Set (Cat. No. 2 158 850) is used to determine the level of crosstalk by measuring the fluorescence of each individual dye in all detection channels, thereby providing the data for a so-called color compensation file. Factors influencing the relative crosstalk values The emitted fluorescence intensity and spectrum of a fluorescent dye is strongly temperature-dependent. Thus the temperature chosen for data acquisition (fluorescence measurement) during a PCR run influences the crosstalk significantly. This is particularly important for applications where measurements are performed at different temperatures (e.g., during melting curve analysis). During melting curve analysis, continuous measurements of fluorescence are made at slowly increasing temperatures. The measured fluorescence crosstalk is not only dependent on the temperature of data acquisition but also on the specific hardware components of the optical system of each specific instrument. Minor variations in the optical filters, lenses, and dichroic mirrors of each LightCycler Instrument s fluorimeter have an influence Figure 3: Fluorescence excitation and emission spectra of fluorescein, LightCycler-Red 640, and LightCycler-Red 705. 8 BIOCHEMICA No. 2 1999

Figure 4a: without color compensation Figure 4: Temperature-dependent color compensation. on crosstalk values. Therefore, it is necessary to generate and store an instrument-specific color compensation file before starting dual color applications on a LightCycler Instrument. It has been shown that the optimized LC reagents and kits used in the LightCycler PCR or RT-PCR systems do not display a relevant influence on relative crosstalk values. Furthermore, it is shown that different oligonucleotide sequences used for Hybridization Probes do not influence the crosstalk. The 5 coupling chemistry of the LC-Red 640-NHS-ester in combination with commonly used C6-Phosphoramidites (ABI, Glen Research), or the coupling chemistry with LC-Red 705-Phosphoramidite, results in a uniform structure of the linkers between the dye and Figure 4b: with color compensation oligonucleotide for each of the two fluorophores, and thus does not influence relative crosstalk values. In contrast, fluorescein can be coupled to the 3 end of an oligonucleotide using different linker chemistries. The most frequently used are thiourea linkers (FITCderived, for example fluorescein-cpg s from Glen Research or ChemGene) and amide-linkers (fluorescein-nhs-ester-derived, like fluorescein-cpg from BioGenex), or 3 -amino-cpg s that require coupling of fluorescein-nhs-ester after oligo synthesis. Thiourealinked fluorescein oligos display different relative crosstalk values compared to amide-linked fluorescein oligos. Therefore, both linker types are not compatible for quantitative crosstalk correction. For the LightCycler-Color Compensation Set, a fluorescein thiourea linker is used. As a result, it is Vial Label Contents and use 1 Blank standard buffer excluding fluorophores to determine the standard buffer fluorescence 2 Fluorescein Calibrator standard oligonucleotide, fluorescein-labeled at the 3' end [0.3 µm] to determine the crosstalk from fluorescein (channel 1 fluorophore), into channels 2 and 3 3 LightCycler-Red 640 Calibrator standard oligonucleotide, LC-Red 640-labeled at the 3' end [1 µm] to determine the crosstalk from LC-Red 640 (channel 2 fluorophore), into channels 1 and 3 4 LightCycler-Red 705 Calibrator standard oligonucleotide, LC-Red 705-labeled at the 3' end [1 µm] to determine the crosstalk from LC-Red 705 (channel 3 fluorophore), into channels 1 and 2 Table 1: Components of the LightCycler-Color Compensation Set 9

necessary that user-specific Hybridization Probes are also fluorescein-labeled with a thiourea-linker when performing dual color applications. When ordering Hybridization Probes from licensed oligonucleotide suppliers (e.g., GENSET, page 20), the fluoresceinlabeled Hybridization Probe will automatically be labeled using the thiourea-linker. Generation and use of a color compensation file (ccc file) An instrument-specific color compensation file can be created that subsequently can be used for color compensation of the raw data from different dual color experiments and applications. The color compensation file is generated using the LightCycler-Color Compensation Set. The set consists of three calibrators to determine the relative contribution of each individual fluorophore to the total fluorescence observed in the 3 detection channels (Table 1). This relative contribution does not depend on the FRET process and thus does not depend on the presence of a hybridization target. In addition, the set provides a standard buffer to determine the general influence of non-dye components on the crosstalk. The procedure to set up and save a color compensation file takes approximately 45 minutes, including the LightCycler Instrument calibration run, with only 5 minutes hands-on-time. All 4 components of the set Figure 5a: without color compensation Figure 5b: with color compensation Figure 5: Color compensation of a dual color duplex PCR experiment. Target sequences within the GAPDH-gene and the Cyclophilin A-gene were either amplified simultaneously [1: 10 7 copies each; 2: 10 6 copies each; 3: 10 5 copies each] or as a single target [4:10 9 copies GAPDH; 5: 10 8 copies Cyclophilin A]. GAPDH is detected with a fluorescein/lc-red 640 probe pair and Cyclophilin A with a fluorescein/lc-red 705 probe pair. Figure 5a: raw data from channels 2 and 3. Figure 5b: data after color compensation. 10 BIOCHEMICA No. 2 1999

are ready-to-use solutions. Each is pipetted into a single capillary and placed into the LightCycler Instrument. The experimental protocol consists of 4 program steps: heating step, cycling step, temperature gradient step (with continuous data acquisition), and cooling step. For detailed conditions, see the pack insert of the LightCycler-Color Compensation Set or the Light- Cycler Operator s Manual. Applications The color compensation file must be used for any application where two reporter dyes are used simultaneously. This includes: Use of internal controls (e.g., to exclude PCR inhibition). After the run, the data are stored as a color compensation file. Detection of more than one target sequence in a single sample. In subsequent dual color experiments, the color compensation file can either be used to compensate raw fluorescence data on-line during a LightCycler Instrument run, or after the run during data analysis. Detection of complex mutations. See the article Mutation Detection Using Multi-color Detection of the LightCycler System on page 12 of this Biochemica issue. Figure 4 illustrates the temperature-dependent contribution of all fluorophores to the fluorescence in each channel of the LightCycler Instrument. Figure 4a demonstrates the fluorescence raw data that are measured in each channel during the temperature gradient step while generating a color compensation file. Figure 4b shows the same data after color compensation using a previously stored color compensation file. It is obvious that after color compensation, each channel displays specifically the signal from the channel-specific fluorophore, whereas non-specific signals are compensated. Development formats of even more precise quantification using internal standards. Measurement of a known number of copies of a standard sequence, and simultaneous measurement of the copy number of a target sequence in the same capillary, guarantees identical PCR conditions for both amplicons. Appropriate software for the evaluation of the results of these types of experiments is under development. Figure 5 demonstrates the application of color compensation to a duplex PCR experiment using two Hybridization Probe pairs for the detection of amplicons from two different equimolar targets. Target 1 amplicons are detected using a fluorescein LC-Red 640-labeled probe pair and target 2 amplicons are detected using a fluorescein LC-Red 705-labeled probe pair. In addition to the duplex PCR, both targets are also amplified separately in different capillaries. Raw data and color compensated data are shown for channels 2 and 3 for all three capillaries. After color compensation, the contributions of the nonspecific dyes to the fluorescence signals are eliminated, as clearly demonstrated in the uniplex reactions. 11