Hello Everyone
I have been working on 14 different virus strains to be detected utilizing TaqMan Probes. Although I get good results in Singleplex assays with the almost same Ct for all samples, in multiplex assays 3 types have higher CTs (more than 5 cycles), Do u have any suggestions to overcome this issue? the plasmid copy number, primer-probe concentration, and qPCR probe Master mix are the same.
It might be worth taking these 3 primer sets and running them in all combinations,1+2,1+3,2+3 to see if you are getting primer dimer which removes primer very quickly and can result in low yield pcr. If there is a lot of dimer then redesign of primer or use of a hot start enzyme may help
Thanks for your time and consideration, I already checked primer dimer by bioanformatic tools, I dont see much primer dimer and even after running real-time I run all the reactions on Agarose gel. As with a hot start polymerase I have only problem with with 3 primers, I'm afraid that all reactions changes in thier Cts. But I will try it. I'm thinking about changing these primers but don't know what is exactly wrong with these 3 primers.
often you do not see much primer dimer because the amount of EtBr trapped by dsDNA depends on the length of the dna and PD are very short so if you do see any then there is quite a lot of it. If you are sure that there is very little PD then increasing the amount of these 3 primer sets may shift the equilibrium to produce more pcr product. Of course also you must check that there are no polymorphisms at, or near the 3' end of any of these 6 primers which would decrease the efficiency of these pcrs but this is not likely if the dna used in the multiplex reaction is the same dna as in the single plex setup pcrs. Possibly if any of these primers have a motif looking like GGGG then the increased salt concentration of the added other primers may be favouring a G quadruplex causing a hairpin in the primer and lowered binding and yield in which case dmso would help the reaction although it would need teting against the other primers which is work that you do not need whn things are nearly working so well
Dear @Paul Rutland
I really appreciate your help.
I have 14 different templates with same concentration. The products are around 100bp. I didn't think that PD migt be an issues not only because I didn't see PD by bioanforamtic tools, but also after running Agarose Gel I saw a very weak PD from reactions with similar Cts in which they consider as Working-Well samples.
I will try DMSO, BSA, and PEG to see if any changes will occur.
That is a good point about seeing the same level of PD in the other primer sets Ahmad Nasiri Good luck with the pcr enhancers and if these fail it may still be worth trying increased amount of primer for the 3 poorly performing sets
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