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

(For additional product information, click on the column headers below.)

  SYBR® Green I Dual-Labeled Probes Dual-Labeled LNA® 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     • •    

*LNA can be incorporated into the probes for improved specificity.

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.

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
Texas Red® 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


Reporters and Instrument Compatibility Table

Platform SYBR
Green I
FAM HEX JOE ROX TET Cya-
nine 3
Cya-
nine 5
TAMRA Texas
Red
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

 

Materials