Choosing the Right Custom qPCR Probe

There is a wide selection of possible custom probes that can be used for qPCR, and each has advantages for different applications. The summary below highlights the applications of commonly used custom qPCR probes.

Application Reference Guide

Reporters, Quenchers, and Instrument Compatibility

Modern qPCR platforms typically have multiple detection channels enabling flexibility in the choice of probe labels. It is important to select the fluorescent labels which are compatible with the detection channels for the qPCR instrument and to ensure the correct filter settings or detection calibration for the instrument. The Reporters and Instrument Compatibility table lists a selection of some of the most widely used qPCR platforms and indicates which fluorescent labels may be used. Please note that not all labels are listed and many alternative reporters are available. For information on the use of non-standard labels with these platforms, contact your local technical service professional.

Dye Substitutes

Several qPCR instruments utilize proprietary dyes which are not generally available commercially, such as VIC^® and NED™. When seeking dye alternatives, the following criteria are important:

• The excitation and detection wavelength are compatible with the instrument light source and detection system
• For probes, the quencher effectively absorbs light at the emission wavelength of the reporter
• The higher the extinction coefficient the brighter the dye, which contributes to sensitive detection
• When using multiple dyes (multiplex) the excitation and emission wavelengths of each dye must be independent to avoid cross talk

Quenchers

Quenching molecules are typically placed at the 3' end of single molecule probes such as Dual-Labeled Probes, Molecular Beacons and Scorpions Probes. Quenchers may be fluorescent (TAMRA™) or nonfluorescent molecules (DABCYL, Black Hole Quencher™ (BHQ™). For optimal performance, the quencher's absorbance spectrum should match the reporter’s emission spectrum as closely as possible. Recommended reporter/quencher combinations can be found in the Spectral Properties table.