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

Table 1. *Locked Nucleic Acid can be incorporated into the PCR Primers or qPCR probes for improved specificity. Locked Nucleic Acid in oligonucleotides other than qPCR probes is available in all countries except for the United States.

	SYBR® Green I*	Dual-Labeled Probes	Dual-Labeled Locked Nucleic Acid Probes	Molecular Beacons*	LightCycler® Probes*	Scorpions® Probes*
APPLICATION
Mass screening	• •
Microarray validation	• •	•
Multiple target genes / few samples	•	•
SNP detection			• •	•	•	• •
Allele discrimination			• •	•	•	• •
Pathogen detection	•	•	• •	•	•	• •
Multiplexing		• •	• •	• •	•	• •
Viral load quantification		•	• •	•	•	• •
Gene expression analysis	•	• •	• •	• •	• •	• •
Gene copy determination		•	• •	•	•	• •
Endpoint genotyping				• •		• •
in vitro quantification or detection			•	• •

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.

Table 2. Spectral Properties Table

Dye	Max. EX (nm)	Max. EM (nm)	Compatible Quencher
6-FAM™	495	520	BHQ™-1, TAMRA
JOE™	529	555	BHQ™-1, TAMRA
TET™	521	536	BHQ™-1, TAMRA
Cal Fluor® Gold 5401	522	544	BHQ™-1
HEX™2	535	556	BHQ™-1, TAMRA
Cal Fluor Orange 5602	538	559	BHQ™-1
TAMRA	557	583	BHQ™-2
Cyanine 3	549	566	BHQ™-2
Quasar® 5703	548	566	BHQ™-2
Cal Fluor Red 5904	569	591	BHQ™-2
ROX™	586	610	BHQ™-2
TxRd (Sulforhodamine 101-X)	597	616	BHQ™-2
Cyanine 5	646	669	BHQ™-3
Quasar 6705	647	670	BHQ™-3
Cyanine 5.5	675	694	BHQ™-3
1JOE/TET alternative 2VIC® alternative 3Cyanine 3 alternative 4TAMRA alternative 5Cyanine 5 alternative

Table 3. Reporters and Instrument Compatibility Table

Platform	SYBR Green I	FAM	HEX	JOE	ROX	TET	Cya- nine 3	Cya- nine 5	TAMRA	TxRd	LC Red 640	LC Red 705
ABI 7900HT	•	•	•	•	•	•			•
ABI 7300	•	•	•	•	•				•
ABI 7500	•	•	•	•	•		•	•	•	•
ABI 7700	•	•	•	•		•			•
ABI 7000	•	•	•	•					•
ABI StepOne™	•	•	•	•	•		•		•
ABI StepOnePlus™	•	•	•	•	•		•		•
Bio-Rad iQ™ 5	•	•	•	•	•	•	•		•	•
Bio-Rad Opticon® 2	•	•	•			•			•
Bio-Rad Chromo4™	•	•	•	•	•	•	•	•	•	•
Bio-Rad MyiQ™	•	•
Bio-Rad MiniOpticon™	•	•
Bio-Rad CFX96™	•	•	•	•	•	•	•	•	•	•	•	•
Bio-Rad SFX384™	•	•	•	•	•	•	•	•	•	•	•	•
Agilent Mx4000®	•	•	•	•	•	•	•	•	•	•
Agilent Mx3000P®	•	•	•	•	•	•	•	•	•	•
Agilent Mx3005P®	•	•	•	•	•	•	•	•	•	•
Roche LightCycler®	•	•									•	•
Roche LightCycler 2	•	•	•								•	•
Roche LightCycler 480	•	•	•					•			•	•
Cepheid SmartCycler®	•	•				•	•			•
Cepheid SmartCycler II	•	•				•	•	•		•
Qiagen Rotor-Gene® 6000	•	•		•	•	•	•	•	•	•
Eppendorf Mastercycler® realplex	•	•	•	•	•				•	•
Note: Not all qPCR instruments or fluorophores are listed. Contact the instrument manufacturer for details on compatible fluorophores