quencher position

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Petrusha
Petrusha's picture
quencher position

Hello,
I´ve designed a set of TaqMan probes I´d like to use in qPCR. The length of probes is between 20-30 bp. Do you recommend to place dark quencher on the 3´end of the longest probe or is it better to place it around 20 nt to reduce background? Does the quencher position influence probe´s Tm?

Jitendra Sinha
Jitendra Sinha's picture
Normally if you are using 20

Normally if you are using 20-25 bp long probe it doesn't influence the Tm so much

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Petrusha,
I am not sure what your question is. When you synthesize a double-labeled probe, with a fluorescent dye at the 5' end and a quencher dye (or BHQ) at the 3' end, you simply add these dyes at each end of the probe. If your probes are between 20 and 30 bp there is no reason why you would need to go through the trouble of adding the BHQ anywhere but at the end of the probe (no matter if it is anywhere between 20 and 30 bp long). Adding the BHQ somewhere in the middle of the probe does not make sense as far as I can envision this. 
Also, as Jiten pointed out, the BHQ's position should not influence the Tm of the probe. 
 

Petrusha
Petrusha's picture
Thanks Jiten and Ivan,

Thanks Jiten and Ivan,
anyway I got also different answers and found an article where relocation of the quencher from traditional 3´position to an internal one increased the sensitivity of probe even it´s length was 29 bp  (http://www.ncbi.nlm.nih.gov/pubmed/12581847?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum)