Hi,
As a beginner of this I wanted to be clear about few things regarding fluorescent probes and their dyes.
In one published document I selected below two probes for a duplex qPCR
1). 5’–(FAM)–CAAAgATggACCCCCACCCACg–(BHQ1)–3’
2). 5‘–(YY)–ATgggTTTTTATgATTAgAgTCCCgCAA–(BHQ1) –3‘
in which the 1st one is with FAM as reporter and can be done in our Illumina EcoPCR. But 2nd one is with Yakima yellow, which is not in our system's drop down list.
My question is that, is there an equivalent dye for YY for my particular probe(Sequence unchanged)?
Or is there any alternative for using the probe got synthesized with YY without a YY filter?
Please help..
Yakima Yellow has absorption maximum at 530 nm and emission maximum at 549 nm. You can probably use the TAMRA settings.
Dear adam,
Thanks for response and TAMRA is not in the reporter list,
We have Cy5, FAM, Green, Hex, ROX, VIC in that list. I am confused how to go ahead with this work.
HEX has an absorption maximum at 535 nm and an emission maximum at 556 nm, similar to Yakima Yellow. Similarly, VIC has an absorption maximum at 538 nm and an emission maximum at 554 nm. Either of these should work.
So dear Dr. Adam, which one can be good to opt
whether synthesizing probe with YY and selecting VIC or HEX filter Or synthesizing the probe with VIC or HEX for replacement of YY?
If you have the option of making the probe with one of the dyes that is optimal for your instrument, you should do that.
probe sequence length matters for dye performance, isn't it Dr.?
My first doubt about this matter was that if we change the dyes as we wish, the proximity variations between one dye to other may effect the performance of the method too.
Dr. Adam, one more help I need on this. Could U please suggest
Where can I find the details of proximity of reporters and quenchers and their sensibility or selection criterion as per taqman probe sequence length?
sorry Dr., if my question is invalid but I am thinking in that way.
Have a look at this web site concerning the BHQ quenchers to make the best pairing between fluorophore and quencher. You will probably want to use BHQ2 instead of BHQ1 for the longer wavelength fluorophore. You can pair it with ROX.
http://www.biosearchtech.com/download/brochures/bhqbrochure.pdf
The identity of the fluorophore will not have a significant effect on the distance between it and the quencher, which is primarily determined by the length of the oligo.
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