Unexplainable smear in PCR

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Unexplainable smear in PCR

Postby AmandaC » Mar 24 2017 12:18 am

Hi,

I have been having this issue with my PCR and would appreciate advice on this matter. I was trying to amplify a 600bp region and the PCRs were working very well - I tested over 200 samples and always obtained a nice clean band until one day it suddenly stopped working and I kept getting this long smear, even in the no template control. I have changed all my reagents (tried different aliquots of water, new aliquots of primers and also ordered new stocks of primers, tested this with 3 different polymerases (hence three different kits), cleaned out gel tank, changed agarose solutions, different genomic preps) and nothing has worked. Also tried to change PCR cycling conditions and different primer and polymerase concentrations but don’t really see any trend in regards to reduction of the smears. I know the smears are not due to too much DNA being loaded because I have tried loading different concentrations of DNA. Also I am seeing this smear in my no template control. I know that my genomic preps are fine (and reagents should be fine too) as my PCRs work well with other primers; it is just this particular primer that gives the smears! In regards to the smears, sometimes it is a complete smear and sometimes I get a band of the right size but also a smear around it, the no template control is a complete smear.

I have now designed new primers to that region (amplifying a 1.8kb region) and at first it was working fine (with no or little smears, no smears in no template control) but after a few PCRs, the smears have returned (even though I have not changed anything)! Again I’ve tested it with different polymerases and water control but am getting smears for all. I’m a bit stumped and would really appreciate any sort of advice. It does seem like it is the primers that is the issue. I have not tried any touchdown PCR or hotstart polymerases so am not sure if that would help..?

Many thanks!!!
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Re: Unexplainable smear in PCR

Postby r.rosati » Mar 24 2017 1:05 pm

Hello Amanda!
How does the smear look like (could you upload an image?) Is it similar to a concatemer? Is there any repetitive or palyndromic sequence near one of the ends of the product?

I saw from your thread on Reddit that you used autoclaved water. Autoclaved water is sterile but it might not be clean, depending on how clean the interior of the autoclave is.

You mention that you used several batches of primers: did they work at start, and then stop working? Primer degradation might explain your results. If the primers work at start, then how long does it take for a new primer batch to stop working?

As testing goes, I would stick with the kit (polymerase and buffer) that were working. I think switching kits might unnecessarily add variables to your problem.
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Re: Unexplainable smear in PCR

Postby relaxin » Mar 24 2017 3:10 pm

Your primers partially annealed to contaminating DNA in the water and started a nonspecific amplification. Hot-start polymerase may reduce this nonspecific amplification. It is worthy of the higher price. I always use hot-start polymerase or use a wax barrier to separate the polymerase from the primer + template.
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Re: Unexplainable smear in PCR

Postby AmandaC » Mar 26 2017 11:02 pm

Thanks for the advice! Really appreciate it!

I've uploaded some images! For some PCRs I get complete smears, occasionally for some PCRs I get the band of the right size with smears - I could use the same sample on two different days using same PCR reagents, conditions etc and one day I get complete smears and the other day I get some amplification with smears (still not good enough as I need to digest & sequence the PCR products and the smears make that not possible)

r.rosati - thank you! I'm not sure if it is similar to a concatemer, but looks quite similar to that photo! There are a lot of AAAs in the sequence, including at the ends of the product. Primers themselves have 55% GC content. I tried commercial bought nuclease free water in a PCR last week - smears in the no template control (master mix only). Interestingly I had one control where I added primers, polymerase etc but did not make it up to required volume with water and there was no smear in that one....

Primers - 1st batch stopped working when I ran out of my 10uM working solution and made up new 10uM primers from my 100uM stock solution so I thought it must have degraded; second batch and third batch (ordered them from two different companies) - saw smears from the start with the two new primers. Now interestingly I had a set of the primers in a different lab in a different location (the primers were older than any of those other three) so I went back to the lab and did a PCR there with the primers, reagents and genomic preps that I had in the lab there - worked beautifully so I brought over the primers, PCR reagents and water from the old lab to the new lab - at first didn't get any smears using those (although the PCR was not working well for all samples; didn't get any amplification for all samples) but after a few tries, started seeing smears again.

I designed new primers - first couple of PCRs was fine and then after a few weeks, smears with these new primers! I am not sure if the primers are binding to some sort of contaminant or forming some sort of adduct with itself? Could it be something in that lab? But I have tried different pipettes, different tubes, different Thermocyclers, different gel tanks and running buffer, even use a different bench that no one used previously, I can't figure out where the contaminant is coming from.

relaxin - thanks! I believe one of the polymerases that I was using is a hotstart polymerase, but it is also a ReadyMix (polymerase + buffer + dNTPs + MgCl2 etc all in one) (KAPA Robust HotStart ReadyMix), convenient but if it is not working, can't figure out if it is one of the components.
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Re: Unexplainable smear in PCR

Postby relaxin » Mar 27 2017 5:00 pm

There are other hot-start polymerases that come with buffer only. It is better to prepare your own master mix than to use the ready-mix.
When you thaw out buffer and reagents, let them thaw out completely and mix well before pipetting.
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Re: Unexplainable smear in PCR

Postby r.rosati » Mar 28 2017 1:21 pm

Hello Amanda,
in general, with smears like you experience, I would have some advice like trying to raise the annealing temperature, using a HotStart polymerase as relaxin suggested, decreasing the number of cycles, reducing the extension time in the PCR program, and possibly decreasing the amount of template.
However, your PCR was working at first, and with the same conditions where it's now failing to amplify, which invalids the idea that the PCR needs optimization of any sort.
I'm afraid contamination is right now the most likely option... any chance that some of this smear is making its way into the pre-PCR area? Are you both mounting the PCR and purifying PCR products on the same bench? Pipettes being used both pre- and post-PCR?
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Re: Unexplainable smear in PCR

Postby mdfenko » Mar 29 2017 11:36 am

based on your description of events (especially preparation of samples and blanks), it appears that your source of contamination is the water used when preparing the reactions.

try using fresh di water (eg milli-q) each time you prepare reactions.
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