I'm designing a triplex real time PCR multiplex using Taqman probes. All three sets of primers and probes work perfectly for each of their targets in monoplex. When the three primer and probe sets are mixed, two of them work perfectly but one fails to amplify anything at all even from DNA targets that are exactly the same preparations that amplify fine with the monoplex.
It's not like the amplification of the third target is reduced, it's completely eliminated, even with templates that only include the target for this third primer/probe combination.
Any ideas on how to work out what is happening?
There is a myriad of reasons. First all three amplicons need to be close to the same size, GC content, no homology between primers or other amplification products. Anneal with hot start moving fast. Use small reaction volumes 10ul, keeping a high annealing temp around 60C, amplicon size not more than two hundred bps. Do two at a time before going to three. Mg2+ 1-2mn, also do titrations for primers. 30 seconds at any of the cycles is over kill, except your initial denaturation. Have a total %GC so know if you may need to bias dNTPs. If still having problems focus on the one being inhibited and see if increasing target for that helps. Will answer if a total inhibition or a bias. May need to shorten inhibited amplicon. The flip side is lengthen the most robust, or even put a small stem loop near 5" end of primer.
If none of this help, then can get fancier. Make sure all primers and amplification products are blasted against each other and the rest of the genome.
Good Luck.
This may be incredibly obvious (I apologize if so) but are you sure that your probes don't have overlapping emission and absorbance spectra? For example FAM/TAMRA interfere; HEX dye is emits at a wavelength nearly identical to a number of other probes (VIC/JOE, ect). If you assay performs very well individually, make sure that dyes you have chosen do not interfere with one another on your qPCR platform.
Our diagnostic lab. do not like multiplex real time pcr due to when making multiplex the sensitivity is decreased due to competition on reagents, therefore, it is better to not exert a lot of efforts and time in optimization step. if you examine many samples with different PCR protocol, it is preferred to work with smart cycler that give you chance to make separate pcr protocol in each well
is all three targets are in the same gene ?
check your primer design...
if your target sequence are closer ..check third primer-s (that one fails to amplify ) is between other two primers amplifying sequence ..that might be the problem
good luck..
Thank you for all your replies.
We need to make this a multiplex due to the cost implications of doing this routinely.
All amplicons are of a similar small size.
GC looks comparable.
The inhibited amplicon can't be any shorter.
Reaction volume is small and uses hot start and the standard fast PCR cycle.
Increasing or even decreasing target three template has absolutely no effect.
Primers, probes and amplification products show no problematic complimentary.
Target one with target three in duplex works fine.
Target two with target three in duplex works fine.
Excitation and emission spectra look fine ( VIC, FAM and Cy5).
All the targets are for totally different genes on totally different species.
I'm at a total loss!
We are in the same boat. In my case, I will try to optimize the concentration of primers and probes first. The thing that I bear in mind is concentration of primer affects Rn values while concentration of probe affects Ct. I plan to optimize primers and probes until I get high Rn and low Ct. The second thing that we should be aware of is the prevalence among genes or species. A higher prevalent gene or species should reduce the concentration of primers and probe. This is my plan that I will try first. If you have any suggestion or any previous trial that you have made, please share it. I believe it would benefit to both of us.
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