peculiar PCR inhibition - (Oct/21/2005 )
Hi,
I'm working on DNA extracted from museum specimens fixed in formalin and preserved in EtOH. I have DNA, but my PCR gives only primer dimers. I use fresh material extracted exactly the same way as a positive control. When I mix the two templates (fresh and preserved) increasing the amount of preserved (even when keeping fresh the same) decreases the band intensity. This indicates inhibition, but the primer dimers are still there so the Taq is working. I originally blamed structural changes in the DNA preventing binding by Taq, but there must be something else in the extract that inhibits amplification...but not of primer dimers. My product is <200bp. Does anyone have any ideas? Because of the preservation I am quite sure it is not RNA.
What is your positive control? Is it possible that one or the other (or both) of your primers match the positive control template but not your museum specimen template? Even a single base mismatch at the 3' end of either primer can cause the PCR to fail.
Also, as a side note, if your primers are designed correctly, you should not be getting primer dimers (caused by the primers annealing to themselves rather than to your template)...
My positive control is the same species, same tissue (Haplochromis - cichlid- liver), but a freshly sacrificed specimen. I know the primer dimers are not desirable, but these are published primers that I know work on fresh material in spite of the dimers, and provide useful information for species discrimination.
Well, the primer mismatch of ancient (is it ancient?) DNA theory still might be applicable, but let's set that aside for the moment...
The other difference between your control DNA and your experimental DNA is that your experimental DNA is (initially at least) stored in formalin and ethanol.
How are you cleaning up these specimens in preparation for PCR?
I know even trace amounts of EtOH can inhibit PCR, and though I'm not sure about formaldehyde, I suspect it would be inhibitory also...
The preserved tissue goes through several washes in a buffer designed to bind formaldehyde over about 4 days. The tissue is digested with proteinase K and then the DNA is purified with the qiagen DNeasy kit. I use a nanodrop to spec the DNA and find low concentrations, but not the values I would expect with contaminating ethanol - I've also spec'd the different steps in the DNeasy kit and the ethanol is readily identifiable. The DNA also remains in the wells of a gel - ie: not floating the way it will with residual ethanol.
The preserved fish are only 30years old, and these primers amplify microsatellites not only from my species, but are conserved in the species flock, so they should match...
Hmmm... This is a peculiar problem. I guess my next step would be to recover DNA from some of your freshly-killed specimen the same way, all the way through, and see if the PCR works on it. That will rule out your method of DNA recovery as contributing to the PCR failure.
If, after recovering DNA from your control specimins by that method, the PCR fails on that template (where it worked previously), then your recovery method is contributing something to the final DNA that is causing the PCR to fail.
If the control template thus acquired still works in PCR, then we'll look to the primers or PCR conditions as the source of the difficulty...
The freshly killed samples are treated exactly the same way the entire way through; digestion, extraction, pcr in parallel- and the fresh works consistently. A strong band is always visible in the fresh material, and not in the preserved. when the two are mixed, the signal decreases. c'est bizarre...
the pcr conditions have been played with, ie: template amounts, BSA, cycling, annealing temp, touchdown...and always works on fresh - the primers have been used by others as well on the same program and always work on fresh fish, conserved over different species. i'm also using a variety of specimens to rule out a random fluke of sequence mutation.
my reasoning is that something from the preserved tissue is not being removed by the extraction, something that inhibits binding of the taq to the target dna...but does not inhibit primer dimer formation.
Sounds that way, doesn't it? Of course, we're looking at inconclusive evidence -- just because the primers match everything you've tried still does not rule out the possibility that one or the other of them does not match your older template.
At this point, I would try different primers, even if they're just a bit shorter but otherwise identical to the originals, to overcome a possible point mutation in just the wrong spot (3' on the primer). I understand your reluctance to do that, though.
How about trying to clean up the DNA further with a phenol:chloroform extraction, and ethanol precipitation?
Do you have enough DNA to try sequencing it with your primers? If one or the other primers is not working with your preserved template, it'll fail to produce a sequence (of course if neither primer produces a sequence, we've accomplished nothing).
If your inhibitor binds to the DNA infrequently then it might be binding enough to prevent amplification of your product but not so frequently that it doesn't prevent primer dimer formation. I have seen this effect before where I have two products in a PCR and the large one can be inhibited while the smaller amplifies fine.
Daniel
Higher quality DNA sequencing traces
At this point, I would try different primers, even if they're just a bit shorter but otherwise identical to the originals, to overcome a possible point mutation in just the wrong spot (3' on the primer). I understand your reluctance to do that, though.
I understand your reasoning - but looks like i didnt mention i'm actually getting the same results using 4 primer sets. All different microsatellites. All published in the same paper - but that should rule out any weird mismatches occurring with any one primer.
Daniel - point taken.
I will try to do more cleanup...phenol/chloro w/ethanol precipitation has always been my standby, any other suggestions? i know most kits are for post-pcr, not pre.