RT-PCR Controls (no DNA) show weird amplification - Complicated problem- urgent help needed! (Apr/12/2007 )
I've been using RT-PCR using SYBR Green to detect bacterial genomic DNA with primers targetting single-copy virulence genes, but all my controls (no DNA whatsoever) have been showing amplification no matter what I do. When I plot the melting peaks, the control always has a single large peak (83 deg) only a few degrees away from the melting peak of the product in the samples containing DNA (79 deg). I've checked my primers for cross-complementarity and they do actually have a tendency to form primer-dimers, which would of course explain the product in the control. But what concerns me is that the peak is quite large for a primer dimer, and it appears only a few deg away from the peak in the sample with DNA, and the ampliciation curve shows that it starts being produced (i.e. fluorescing) only a few cycles after the product in the samples with DNA.
And when I do dilution series (consecutive dilutions in water from 1 to 1:100,000), the dilutions always have two peaks: the product peak (79) and the mysterious control peak (83), but of different heights of each for every dilution. Even though the undiluted DNA template only gives a single product.
When I run the RT-PCR products on a gel I get a perfect single band with my undiluted DNA template, and several messy bands with my control and different DNA dilutions. I've attached a picture of the gel, showing the products of RT-PCR rxn. Well #9 is with the undiluted DNA template, well #10 is the control (no DNA), #12 & 13 are 2 different dilutions of the template, showing 2 bands and smears at the top.
I've repeated this experiment exactly 3 times with exactly the same inconsistent results, with the different dilutions flip-flopping back and forth between the 2 peaks. I just don't get it. All the RT-PCR experts around here are just as stumped as I am. Any help would be very very appreciated!
okay so not necessarily correct but an idea I had...
so I think (based on experience not necessarily empirical evidence) that sometimes aberrant primer binding is dependant on the concentration of the template. So that band in my opinion is too large to be primer dimers, most likely it is a mispriming location for the primer pairs. When there is alot of the correct sequence around the primers are busy binding to the right thing so you don't get as much aberrant product formed in the early cycles however at lower template concentrations there are alot of primers "looking for something to do" sometimes they make primer dimers sometimes they will bind to the template and misprime an amplification product. Once this has happened the "correct" primer sequence is included in the aberrant product and it wil amplify at a similar efficiency to your correct product (of course it is longer etc so probably somewhat lower efficiency but should be similar because the exact primer sequence is now incorporated into the aberrant target.)
Ok so what does this mean experimentally? I would use a higher temperature for annealing, maybe you can prevent mispriming in the first place. you can also do touchdown PCR to enrich for the correct product first, but dont know if that will work well for sybergreen quantitation. You should blast your primers and see if there are any mismatched primers that give the same product size you see (ie look for an aberrant target that is in both blast searches and see if the size/orientation match) if you find the sequence giving problems you can re-design the primers to avoid or just redesign primers peroid...
hope this helps you and good luck!
I would second beccaf22. It could also help to decrease primer concentration.
Keep in mind too that Taq polymerase is commercially synthesized in E. coli, and that since Taq is a DNA-binding enzyme in addition to a polymerase, it's almost impossible to obtain Taq mixes that are entirely free of bacterial DNA. So... if your primers have any cross-homology to genes in E. coli, one of your peaks (esp. in your no-template-controls) might be coming from the inherent DNA "contamination" in the polymerase mix itself.
You might also want to optimize your primers with gradient PCR just in case you're picking up non-specific products simply by using too low an annealing temp.
Thanks for the suggestions everyone, I've also realised that I've been setting my cycle number far too high (45 cycles) and that I haven't been adding any water to the NTC to substitute the DNA volume. I'm going to try increasing my annealing temperature along with the control volume/cycle number corrections...it's too bad the Light cycler I'm using doesn't have a temperature gradient option.
Will keep you posted on the results!