Selection of Fluorophore Quencher Pairs

With the development of new nucleic acid synthesis chemistries and the introduction of new spectrofluorometric thermal cyclers, many manufactures have introduced new types of fluorescent hybridization probes and they promote them as being solely compatible with their products. Fortunately, most types of fluorescent hybridization probes are compatible with all available instruments. Many newly developed types of fluorescent probes are derived from, or utilize mechanisms that are similar to those used by adjacent probes, 5'-nuclease probes, molecular beacon probes, or strand-displacement probes. The end user should therefore consider what spectrofluorometric thermal cycler platform is available, whether the assays that they perform require multiplexing or high sample throughput, and whether the type of fluorescent hybridization probe they choose provides the specificity and sensitivity required to meet the goals of their research or clinical diagnostic applications. Table 2 provides an overview of the spectrofluorometric thermal cyclers currently available. The table specifies the type of excitation source that is utilized in each instrument, which fluorophores are compatible with the optics of each instruments, how many different fluorophores can be analyzed in a single assay (multiplex capability), how many samples can be analyzed simultaneously, and which type of hybridization probes can be used with each instrument. The fluorophores listed in the table can be replaced with alternative fluorophores (listed in Table 3) that exhibit similar excitation and emission spectra and are available from different vendors. Table 4 provides a list of available quencher moieties.

Table 2

Specifications of Spectrofluorometric Thermal Cyclers

Excitation

Multiplex

Sample

Hybridization probe

Company

Model

source

Fluorophore choice"

capability

capacity

compatibility

Applied Biosystems

PRISM® 7000

THL

FAM, TET, TMR, and Texas Red

4 targets

96 wells

All types, except adjacent probes

Applied Biosystems

7300 real-time

THL

FAM, TET, TMR, and Texas Red

4 targets

96 wells

All types, except adjacent probes

PCR system

Applied Biosystems

7500 real-time

THL

FAM, TET, TMR, Texas Red,

5 targets

96 wells

All types, except adjacent probes

PCR system

and Cy5

Applied Biosystems

PRISM 7700

ABLL

FAM, TET, HEX, TMR, ROX,

6 targets

96 wells

All types

and Texas Red

Applied Biosystems

PRISM 7900HT

ABLL

FAM, TET, HEX, TMR, ROX,

6 targets

384 wells

All types

and Texas Red

Bio-Rad

iCycler® IQ

THL

FAM, HEX, Texas Red, and Cy5

4 targets

96 wells

All types

Cepheid

SmartCycler® II

LEDs

FAM, Cy3, Texas Red, and Cy5

4 targets

16 units'*

All types, except adjacent probes

Corbett Research

Rotor-Gene™ 3000

LEDs

FAM, HEX, Texas Red, and Cy5

4 targets

72 wells"

All types, except adjacent probes

Idaho Technologies

R.A.P.I.D.

LED

FAM, LC Red 640,

3 targets

32 wells"

Adjacent probes and wavelength-shifting

and LC Red 705

molecular beacons

MJ Research

Chromo 4™

LEDs

FAM, TMR, Texas Red, and Cy5

4 targets

96 wells

All types, except adjacent probes

Roche Applied Science

LightCycler

LED

FAM, LC Red 640,

3 targets

32 wells"

Adjacent probes and wavelength-shifting

and LC Red 705

molecular beacons

Roche Applied Science

LightCycler 2.0

LED

FAM, HEX, LC Red 610,

6 targets

32 wells"

Adjacent probes and wavelength-shifting

LC Red 640, LC Red 670,

molecular beacons

and LC Red 705

Stratagene

Mx3000P®

THL

FAM, TMR, Texas Red, and Cy5

4 targets

96 wells

All types, except adjacent probes

Stratagene

Mx4000®

THL

FAM, TMR, Texas Red, and Cy5

4 targets

96 wells

All types, except adjacent probes

PCR, polymerase chain reaction; THL, Tungsten-halogen lamp; ABLL, Argon blue-light laser; LED, light-emitting diode. "Refer to Table 3 for alternative fluorophore choices. sEach unit is independent programmable. cRapid cycle capabilities.

Table 3

Fluorophore Labels for Fluorescent Hybridization Probes

Table 3

Fluorophore Labels for Fluorescent Hybridization Probes

Excitation

Emission

Fluorophore

Alternative fluorophore

(nm)

(nm)

FAM

495

515

TET

CAL Fluor Gold 540"

525

540

HEX

JOE, VIC6, CAL Fluor Orange 560"

535

555

Cy3c

NED6, Quasar 570", Oyster 556d

550

570

TMR

CAL Fluor Red 590"

555

575

ROX

LC Red 610e, CAL Fluor Red 610"

575

605

Texas Red

LC Red 610e, CAL Fluor Red 610"

585

605

LC Red 640e

CAL Fluor Red 635"

625

640

Cy5c

LC Red 670e, Quasar 670", Oyster 645d

650

670

LC Red 705e

Cy5.5c

680

710

"CAL and Quasar fluorophores are available from Biosearch Technologies. 6VIC and NED are available from Applied Biosystems. cCy dyes are available from Amersham Biosciences. ^Oyster fluorophores are available from Integrated DNA Technologies. eLC (LightCycler) fluorophores are available from Roche Applied Science.

"CAL and Quasar fluorophores are available from Biosearch Technologies. 6VIC and NED are available from Applied Biosystems. cCy dyes are available from Amersham Biosciences. ^Oyster fluorophores are available from Integrated DNA Technologies. eLC (LightCycler) fluorophores are available from Roche Applied Science.

The following guidelines can be followed in choosing the appropriate fluorophore-quencher combinations for the different types of fluorescent hybridization probes and spectrofluorometric thermal cyclers:

1. Based on the spectrofluorometric thermal cycler platform that is available, choose appropriate fluorophore labels that can be excited and detected by the optics of the instrument. Instruments equipped with an Argon blue-light laser are optimal for excitation of fluorophores with an excitation wavelength between 500 and 540 nm; however, fluorophores with a longer excitation maximum are less well, or not at all, excited by this light source. Instruments with a white light source, such as a Tungsten-halogen lamp, use filters for excitation and emission, and are able to excite and detect fluorophores with an excitation and emission wavelength between 400 and 700 nm, with the same efficiency. This is also the case for instruments that use light emitting diodes as excitation source and emission filters for the detection of a wide range of fluorophores.

2. If the assay is designed to detect one target DNA sequence and only one fluores cent hybridization probe will be used, then FAM, TET, or HEX (or one of their alternatives listed in Table 3) will be a good fluorophore to label the probe. These fluorophores can be excited and detected on all available spectrofluorometric thermal cyclers. In addition, because of the availability of phosphoramidites de rivatives of these fluorophores and the availability of quencher-linked control-

pore glass columns, fluorescent hybridization probes with these labels can be

Table 4

Quencher Labels for Fluorescent Hybridization Probes

Quencher Labels for Fluorescent Hybridization Probes

Table 4

Quencher

Absorption maximum (nm)

DDQ-F

630

Dabcyl

475

Eclipse'

530

Iowa Black FQC

532

BHQ-F

534

QSY-7e

571

BHQ-2d

5S0

DDQ-IF

630

Iowa Black RQC

645

QSY-21e

660

BHQ-3d

670

°DDQ or Deep Dark Quenchers are available from Eurogentec. ^Eclipse quenchers are available from Epoch Biosciences. Towa quenchers are available from Integrated DNA Technologies. dBHQ or Black Hole Quenchers™ are available from Biosearch Technologies. "QSY quenchers are available from Molecular Probes.

°DDQ or Deep Dark Quenchers are available from Eurogentec. ^Eclipse quenchers are available from Epoch Biosciences. Towa quenchers are available from Integrated DNA Technologies. dBHQ or Black Hole Quenchers™ are available from Biosearch Technologies. "QSY quenchers are available from Molecular Probes.

entirely synthesized in an automated DNA synthesis process, with the advantage of relatively less expensive and less labor intensive probe manufacture.

3. If the assay is designed for the detection of two or more target DNA sequences (multiplex amplification assays), and, therefore, two or more fluorescent hybridization probes will be used, choose fluorophores with absorption and emission wavelengths that are well separated from each other (minimal spectral overlap). Most instruments have a choice of excitation and emission filters that minimize the spectral overlap between fluorophores. To the extent that spectral overlap occurs, the instruments are supported by software programs with build-in algorithms to determine the emission contribution from each of the fluorophores present in the amplification reaction. In addition, most instruments have the option to manual calibrate the optics for the fluorophores utilized in the assay to further optimize the determination of emission contribution of each fluorophore.

4. For the design of fluorescent hybridization probes that utilize FRET, fluorophore-quencher pairs that have sufficient spectral overlap should be chosen. Fluorophores with an emission maximum between 500 and 550 nm, such as FAM, TET, and HEX, are best quenched by quenchers with absorption maxima between

450 and 550 nm, such as dabcyl and BHQ-1 (see Table 4 for alternative quencher labels). Fluorophores with an emission maximum above 550 nm, such as rhodamines (including TMR, ROX, and Texas Red) and Cy dyes (including Cy3 and Cy5) are best quenched by quenchers with absorption maxima above 550 nm (including BHQ-2).

5. For the design of fluorescent hybridization probes that utilize contact quenching, any nonfluorescent quencher can serve as a good acceptor of energy from the fluorophore. However, it is our experience that Cy3 and Cy5 are best quenched by the BHQ-1 and BHQ-2 quenchers.

6. Fluorophores exhibit specific quantum yields. Fluorescence quantum yield is a measure of the efficiency with which a fluorophore is able to convert absorbed light to emitted light. Higher quantum yields result in higher fluorescence intensities. Quantum yield is sensitive to changes in pH and temperature. Under most nucleic acid amplification reaction conditions, pH and temperature do not change much and, therefore, the quantum yield will not change significantly. However, in optimizing nucleic acid amplification reactions, quantum yields might change as assay temperatures vary. As a result, lower fluorescence signals at higher temperatures can be falsely interpreted to mean that the fluorescent hybridization probe did not hybridize to its nucleic acid target, when what actually occurred was that the decrease in fluorescence was primarily owing to a lower quantum yield of the fluorophore label. Figure 2 shows the relation between quantum yield and temperature for some of the most common fluorophores. TET, HEX, ROX, and Texas Red do not show a significant change in their quantum yield with increasing temperature, whereas FAM and TMR show a constant moderate decrease in quantum yield with increasing temperature. On the other hand, the Cy dyes, Cy3 and Cy5, show a decrease of almost 70% of their quantum yield at 65°C compared with their quantum yield at room temperature. Consequently, to obtain significant fluorescence signals with hybridization probes labeled with Cy3 and Cy5 fluorophores in assays that are carried out at higher reaction temperatures, higher concentrations of the hybridization probes should be added to the reactions. On the other hand, platforms that are designed for monitoring the fluorescence of Cy dyes, such as Cepheid's SmartCycler® (see Table 2), perform the calibration of the optics for these fluorophores at 60°C to obtain optimal sensitivity.

7. Nucleotides can quench the fluorescence of fluorophores, with guanosine being the most efficient quencher, followed by adenosine, cytidine and thymidine (11). In general, fluorophores with an excitation wavelength between 500 and 550 nm are quenched more efficiently by nucleotides than fluorophores with longer excitation wavelengths. In designing fluorescent hybridization probes, try to avoid placing a fluorophore label directly next to a guanosine, to ensure higher fluorescence signals from the fluorophore.

8. The stabilizing effect of some fluorophore-quencher pairs that interact by contact quenching has important consequences for the design of hybridization probes (10,12). In our experience, hybridization probes labeled with a fluorophore quenched by either BHQ-1 or BHQ-2 show an increase in hybrid melting tem-

Fig. 2. Effect of temperature on the quantum yield of fluorophores. The fluorophores TET, HEX, ROX, and Texas Red do not show a significant change in their quantum yield with increasing temperature. The fluorophores FAM and TMR show a moderate constant decrease in their quantum yield with increasing temperature, whereas the fluorophores Cy3 and Cy5 show a sharp decrease in their quantum yield with increasing temperature.

Fig. 2. Effect of temperature on the quantum yield of fluorophores. The fluorophores TET, HEX, ROX, and Texas Red do not show a significant change in their quantum yield with increasing temperature. The fluorophores FAM and TMR show a moderate constant decrease in their quantum yield with increasing temperature, whereas the fluorophores Cy3 and Cy5 show a sharp decrease in their quantum yield with increasing temperature.

perature of approx 4°C, compared with hybridization probes with the same probe sequence, but labeled with fluorophores quenched by dabcyl. We observed the strongest affinity between the Cy dyes, Cy3 and Cy5, and the Black Hole quenchers, BHQ-1 and BHQ-2.

The ongoing development of fluorescent nucleic acid hybridization probes will result in the introduction of more fluorophore-quencher pairs, with improved biophysical and biochemical properties and wider range of color selection. Together with the introduction of new spectrofluorometric thermal cyclers that allow more fluorophores to be excited and detected simultaneously, this will enable even higher throughput multiplex assays for the sensitive and specific detection of nucleic acids.

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