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Identifying PCR Inhibitors


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#1 Nebulus

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Posted 29 October 2009 - 04:15 PM

Hi,

I'm having some problems with PCR inhibitors in my DNA extracts. I am currently using DNeasy Blood and Tissue Kits. My organisms live in plant phloem so I had suspected there were polysaccharides involved in the inhibition. I have tried adding 2% PVP to the buffer ATL and I have tried post extraction PVPP spin columns to no avail. I cannot dilute my DNA samples (400-600ng/ul) to dilute the inhibitors since I am looking for very small concentrations of prey DNA in my subjects.

Is there any way to determine exactly what inhibitors are present in my samples? It feels like I am running around with my head cut off trying all these purification methods for each type of inhibitor that could be present.

I am only getting about 15% of my samples testing positive compared to 100% during my PCR optimization. I was however diluting my predator's DNA to 25ng/ul during this process, but I am positive my reactions are optimized so that isn't the issue.

Any help would be greatly appreciated since I am in serious trouble of running into some deadlines.

#2 phage434

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Posted 29 October 2009 - 05:13 PM

I would try a CTAB based plant DNA preparation, which will remove most of the phenolic compounds. But remember that diluting your DNA 10x will result in only a 3.3 cycle delay in amplification, so I think you really need to be thinking about dilution as your friend.

#3 Nebulus

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Posted 30 October 2009 - 01:27 PM

Diluting my sample isn't an option for the type of research I am doing, since I am screening the gut contents of my predators for prey DNA. The prey DNA is already in very small quantities and diluting my DNA may give me false negatives.

I've read a lot about the CTAB method, but haven't actually tried it. I am still not sure what class of inhibitors I am getting so I am wary of attempting a new type of DNA extraction technique and wasting time.

I was really hoping there was some type of really good post extraction inhibitor cleanup method, but there seems to be so many different types that claim to be good it is hard to decide.

#4 KimWG

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Posted 09 November 2009 - 11:18 PM

I feel your pain in many, many, many ways -- I'm screening feces from my predators (bats) to look for prey (insect) DNA, and have run into the same problem. While the Qiagen DNeasy kit works well a lot of the time, sometimes, there are just too damn many inhibitors (I've checked by combining template from ones that won't amplify with samples that will -- suddenly, no PCR product.) And like you, dilution hardly ever helps, even with extended cycles in my PCR, the DNA is too low copy/too degraded to get a good balance between dilution of inhibitors and having enough template to work with.

Anyway, FYI -- with these problem samples, I've had good luck re-extracting with the Omega E.Z.N.A. kits (for insects, in my case, but they make lots of kits) -- it's a chloroform extraction for dummies, and samples that wouldn't amp with a Qiagen extraction would suddenly work with the Omega. I realize this may not be an option if you are working with an extremely small amount of tissue in the first place, but the kit isn't that expensive (about $100 for 50 rxns) so you may want to give it a shot.

Of course, I am cheap and lazy so I am cruising around here to see if I can "rescue" my DNeasy extractions instead of re-extracting...

#5 Nebulus

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Posted 10 November 2009 - 01:20 PM

Have you tried only extracting the DNA with the other kit and observing the % success over the DNAeasy kit? How many more samples worked after another extraction with the Omega kit? If it is only a small percentage increase in success it may not be worth it but if it gets me over 90% success (compared to 20%ish) now it is. It also may be worth just only extracting with the Omega kit.

#6 KimWG

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Posted 24 November 2009 - 11:11 PM

Have you tried only extracting the DNA with the other kit and observing the % success over the DNAeasy kit? How many more samples worked after another extraction with the Omega kit? If it is only a small percentage increase in success it may not be worth it but if it gets me over 90% success (compared to 20%ish) now it is. It also may be worth just only extracting with the Omega kit.


Sorry I didn't see your question/reply until now. It depends entirely on the species of bat that made the poop, I don't know if it's differences in their digestion and/or the kinds of insects they eat, but for some, most DNeasy extractions work and for those that don't, 9 times out of 10 the Omega kit works. For other species, it's closer to 25/50. I would guess an overall improvement of 40-60%. Another thing that I have discovered since I replied to your post is BSA, which seems to help out reactions with inhibitors in them. I am having a number of reactions with extractions that had previously failed, or produced weak bands only, produce much better results with a little BSA in them. Extractions that I was going to toss I can now keep.

There's a product I haven't tried that is supposed to remove inhibitors from extractions. It ain't cheap and I contacted the company to see if I could get a sample, and have yet to hear back, but in your case it might be more cost effective than trying another kit. Again, I have no idea how well it would work for you, or me, or anyone...
http://www.mobio.com...ean-up-kit.html

#7 Maddie

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Posted 26 November 2009 - 11:23 AM

I think PTB can help against sugars.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#8 klinmed

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Posted 26 November 2009 - 01:05 PM

Hi,

I'm having some problems with PCR inhibitors in my DNA extracts. I am currently using DNeasy Blood and Tissue Kits. My organisms live in plant phloem so I had suspected there were polysaccharides involved in the inhibition. I have tried adding 2% PVP to the buffer ATL and I have tried post extraction PVPP spin columns to no avail. I cannot dilute my DNA samples (400-600ng/ul) to dilute the inhibitors since I am looking for very small concentrations of prey DNA in my subjects.

Is there any way to determine exactly what inhibitors are present in my samples? It feels like I am running around with my head cut off trying all these purification methods for each type of inhibitor that could be present.

I am only getting about 15% of my samples testing positive compared to 100% during my PCR optimization. I was however diluting my predator's DNA to 25ng/ul during this process, but I am positive my reactions are optimized so that isn't the issue.

Any help would be greatly appreciated since I am in serious trouble of running into some deadlines.


You could try a Taq mutant that has been developed to be very resistant to inhibitors (Omnitaq, www.klentaq.com). For example, it will amplify a single copy gene in a pcr reaction containing 20% blood! It is also resistant to inhibitors in soil, fecal matter etc and is used extensively for forensic work.

See: Kermekchiev, MB et al (2009) NAR 37, doi:10.1093/nar/gkn1055

Hope this helps

#9 Maddie

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Posted 29 December 2009 - 01:59 PM

I am soooo gonna test this OmniTaq. B)
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#10 hanming86

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Posted 30 December 2009 - 08:37 PM

In addition, if u haven't tried, there's this qiagen stool kit with the inclusion of something like a pellet that act as a PCR inhibitor absorber. this probably might help abit against the sample you're encountering.

combine that with omni taq and u might just get what you need.
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#11 Nebulus

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Posted 05 January 2010 - 12:49 PM

I have tried the stool kit. The inhibitX tablet technique reduces the amount of extracted DNA compared to normal DNeasy kits by almost 10 fold.

The best technique I have found so far is using PVPP spin columns..




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