Dear All,
I have a very basic question about the PCR/RT-PCR/qPCR.
The PCR primers and TaqMan probes are designed such that the primers/probe connot bind to the mismatched site but only completely matching sites at the annealing temperatur o when I do the PCR/qPCR, I will choose to adjust the annealing temperature first if there is any non-specific amplification.
But there are a variety of addtives such as DMSO, Betaine, TMAC used in the PCR to enhance the specificity or yield. I checked those related papers, in which they claimed these chemicals could affect the binding affinities between the primers and the template, either by weakening the hydrogen bonds between base pairs or stabilizing the binding of primer and template.
So my question is, now that we can eliminate the nonspecific amplificaiton or enhance the yeild by adjusting annealing temperature, why should we use these additives?
Will these additives change the binding kinetics of the primers/probes?
Bests
Binbin
Additives are mostly needed for a prezide PCR reaction. However, adjusting the annealing temperature may be an option to get more optimal results (perform a gradient PCR with variing annealing temperatures about around the (calculated) annealing temperature offered by providers.
If you are able to run your PCRs without the additives, I do not see any reason to use them. Every reagent in the PCRs have a well-established function, including the additives. If you think that you can run your PCRs without additives, there is no problem. Just make sure that you have the right conditions for a good efficiency. I as well recommend running a gradient PCR first.
Hello
Sometimes you aim to amplify a GC rich (above 65%) region, you would need an additive as enhancer to help you efficiently amplify your fragment.
Another case is in the multiplex reaction. You may have 4 different fragments, varying in size, then perhaps with additives you could have a better results.
There are even more reasons to use enhancers in a PCR reaction such as presence of inhibitors.
Good luck
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