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  orderingQuenchers

Great advances have been made in the area of quenchers over the last decade. The limitations of DABCYL and TAMRA for the development of multiplex systems quickly became apparent. These quenchers were not very efficient with the red-shifted dyes. Two different quenching systems were developed to overcome those limitations, the QSY® series and the Black Hole Quenchers (BHQ)®. Both offer
specific quenchers for different emission ranges. The QSY quenchers are analogs of fluorescein.

Figure 1Figure 1: Quencher Structures

There is now a specific quencher for everything from blue dyes such as Pacific Blue to the extremely red-shifted dyes such as Cy5.5. Although DABCYL has nearly become synonymous with the term "quencher" because of the early Molecular Beacon publications, one can now choose from more specialized quenchers - at little or no added cost. Table 1 will help you determine the appropriate quencher for your application. The more popular dye/quencher combinations are listed in Table 2.

Table 1: Spectral Data of Quenchers
  Abs Max Ext. Coef. Quenching Range
DABCYL 453 nm 32,000 380-530 nm
QSY-35 472 nm 23,500 410-500 nm
BHQ-1 534 nm n/a 480-580 nm
QSY-7 560 nm 92,000 500-600 nm
QSY-9 562 nm 85,000 500-600 nm
BHQ-2 579 nm n/a 550-650 nm
QSY-21 660 nm 89,000 590-720 nm
BHQ-3 672 nm n/a 620-730 nm

The difference between QSY-7 and QSY-9 is water solubility of the conjugate and is of more concern to the chemist than to the end user. We do allow you to specify, however, if your experiments require one or the other. Not only does TriLink offer each of these quenchers, we also offer a wide selection for placement on the oligonucleotide. These quenchers can be placed at the 3' or 5' terminus or internally at any deoxy base, which can be modified at the 5 position. There are some limitations based on the companion fluorescent dye chosen.

Table 2: This dye selection chart provides a quick reference to the dyes we provide, spectral ranges and offers a starting point for quencher-dye pairing.

  Dyes Emmax Absmax Quencher Quidelines*
Pyrene 376 nm 340 nm BHQ-1
Absmax:
534 nm
QSY-35
Absmax:
475 nm
DABCYL
Absmax:
479 nm
7-Methoxycoumarin 410 nm 358 nm
Cascade Blue 410 nm 396 nm
Alexa Fluor® 350 442 nm 346 nm      
7-Aminocoumarin-X 442 nm 353 nm      
Pacific Blue 451 nm 416 nm      
Marina Blue 459 nm 362 nm      
Dimethylaminocoumarin 468 nm 376 nm      
BODIPY 493/503 509 nm 500 nm      
DTAF 516 nm 492 nm        
6-FAM (Fluorescein) 516 nm 496 nm        
Dansyl-X 518 nm 335 nm        
Oregon Green 500 519 nm 499 nm        
Alexa Fluor® 488 519 nm 495 nm        
dT-FAM 520 nm 492 nm        
Oregon Green 488 521 nm 495 nm        
Rhodol Green 523 nm 496 nm        
Oregon Green 514 526 nm 506 nm        
Rhodamine Green-X 528 nm 503 nm        
NBD-X 535 nm 466 nm        
TET 536 nm 521 nm        
Alexa Fluor® 430 541 nm 434 nm   QSY-7 & 9
Absmax:
560 nm
 
2', 4', 5', 7' -Tetrabromosulfonefluorescein 544 nm 529 nm    
6-JOE 548 nm 520 nm    
BODIPY-530/550 551 nm 534 nm      
Alexa Fluor® 532 554 nm 532 nm      
HEX 556 nm 535 nm      
Carboxyrhodamine 6G 557 nm 524 nm BHQ-2
Absmax:
579 nm
   
Alexa Fluor® 555 565 nm 555 nm    
BODIPY 558/568 568 nm 559 nm    
BODIPY 564/570 570 nm 564 nm      
Cy3 570 nm 550 nm      
PyMPO 570 nm 415 nm      
Alexa Fluor® 546 573 nm 556 nm      
TAMRA-X/dT-TAMRA 576 nm 546 nm      
Rhodomaine Red-X 580 nm 560 nm      
BODIPY-576/589 589 nm 576 nm      
BODIPY-581/591 591 nm 581 nm        
Alexa Fluor® 568 603 nm 578 nm        
Texas-Red-X 603 nm 583 nm        
Cy3.5 604 nm 588 nm        
Carboxy-X-Rhodamine (ROX) 605 nm 580 nm        
BODIPY-TR-X 616 nm 588 nm        
Alexa Fluor® 594 617 nm 590 nm        
Alexa Fluor® 633 647 nm 632 nm   QSY-21
Absmax:
5661 nm
 
Alexa Fluor® 647 665 nm 650 nm    
Cy5 670 nm 649 nm    
Carboxynaphthofluorescein 672 nm 602 nm      
Alexa Fluor® 660 690 nm 663 nm      
Cy5.5 694 nm 675 nm      
Alexa Fluor® 680 702 nm 679 nm      
Alexa Fluor® 700 723 nm 702 nm        
Alexa Fluor® 750 775 nm 749 nm        
*There is no guarantee a quencher will be effective in your construct. Quencher-dye efficiencies must be empirically determined in your experimental conditions.

 

 

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