PCR inhibitors in soil DNA - (Nov/20/2007 )
These days I have been doing DNA isolations from soil to quantify bacteria and archaea amoA genes. Nevertheless, when performing the Real-Time PCR reactions I do not obtain the expected PCR products of none of them; I get many peaks instead when looking at the melting curve plot onto the Real-Time PCR machine screen. That is, I am obtaining a lot of unspecific products but not the expected ones and most times I do not get amplicons at all.
I suppose that is due to the high amounts of soil PCR inhibitors present into my DNA samples. I hadn't had such a problem in the previous experiments likely because I was using another type of soil (vertisol), now I am using a very acid soil (oxisol). In order to try to overcome this constraint, I diluted my DNA samples up to 10,000-fold, but it didn't work!!! Besides, I test PCR adjuvants such as BSA, DMSO, and glycerol, but it didn´t work either.
I would like you give me some alternative suggestions to get rid this problem. I would really appreciate them. Thanks ahead of time
Looking forward to hearing from you,
PS: My DNA is ok, I checked it on an agarose gel...
What are you amplifying? Are you using the same set of primers? Can you run your previous working example along with the new ones as a positive control? How are you isolating the DNA? The dilution would have been my first suggestion. You could run a control containing a working PCR example along with some of your newly extracted DNA and test for inhibition. Some things to try: add Mg++, add Betaine, switch enzymes (try Phusion). But the first thing to do is to establish why (and whether) the older samples worked, and what is different this time.
When isolating soil DNA, I get metagenomic DNA; that is, DNA from the entire populations of microorganism present at that moment into the soil. Since I have specif primers I am amplifiying and quantifying by Real-Time PCR bacteria and archaea amoA (ammonia-oxidizing) genes. Of course I used positive controls (soil DNA from an earlier isolation) which always amplify.
In previous experiments with DNA isolated from a different type of soil (vertisol) I used the same primers I am currently evaluating and I didn't have problems with the amplification, I always obtained the expected amoA PCR products. For this new experiment, I only changed the type of soil (oxisol) and I have not get the amplicons so far, I guess due to the PCR inhibitors inherent of this type of soil likely. I tried DNA dilutions (up to 10,000-fold) and PCR adjuvants but it didn;t work.
Please let me know if you come up with new suggestions.
Since you have a working positive control, it is easy to see if inhibition is the problem. Mix varying amounts of your (supposedly) inhibiting DNA into a positive, working PCR reaction and see if it stops working. I'd guess that this is not the problem, but you have an easy way to see. You could try amplifying a known gene from your troublesome sample -- 16S rDNA comes to mind -- and see if it can be amplified. If you get amplifiication from another gene, then perhaps there simply is no copies of the gene you are looking for in your sample.
What method did you use to isolate the DNA? There are several companies offering inhibitor removal solutions. I've used MoBio previously.
If it is a matter of PCR inhibitors like humic acid from the soil, I suggest if you try a simple experiment of running your metgenomic DNA sample (30-50ul volume) on 0.7% low melting agarose gel and run it @ 40V overnight. Cut out the high molecular band and purify it with agarase method (using agarase enzyme) or any relevant method of purifying DNA from LMA gel. Try your PCR from the DNA purified from the Low Melting A.
Phage's suggestion of making sure your sample has the target gene is important and you need to confirm it's the inhibitors that give you problem. Since you already have the vertisol DNA, could you spike some of the DNA into oxisol soil and do the extraction and then PCR to see if it fails? Humic acids bind DNA quite well and gel purification may or may not work.
We have a reagent AquaPrecipi that is designed to remove PCR inhibitors from soil and fecal samples. You simply precipitate the DNA in AquaPrecipi solution and it's good to go for PCR. See the AquaGenomic and AquaPrecipi protocol here. It has been successfully used by other researchers to extract fecal DNA for PCR from field samples (Sahara deserts to Rocky mountain forests) that are rich in PCR inhibitors.
I would love to know if it might work for your samples. Please PM me if you would like to try our AquaPrecipi for PCR inhibitor removal.