DNA overload? - Why some fail and others don't... (Nov/05/2006 )
Hi all, can someone please explain to me exactly how too much template DNA inhibits a PCR amplification. I am screening pools of mosquitoes (approx 10-15) and I have to use a 1:40 dilution of my original DNA extraction elution volume before visible bands appear on the gel. The same thing happens when I perform DNA extractions on isolated bacterial colonies from culture plates using Qiagens DNA mini-kit. On average, I have to carry out a ten-fold dilution on 1 in 15 samples for the PCR (16S rRNA) to work (which it always does on the diluted template).
So, my PCR's are working fine, I'm just curious for a scientific answer to the DNA overload phenomena.
Cheers
Muncher
Hi,
Not sure if yours is the case of DNA inhibiting PCR or that, the residual extraction buffer component(s) in the final DNA solution that's inhibiting your PCR.
Since you're working with mosquito DNA, I think the DNA yield shouldn't be too much to inhibit the PCR?
It's my presumption that, by diluting the DNA sol, you'd diluted the probable cause of inhibition to your PCR, but still maintaining the DNA quantity detectable by PCR amplification.
Not sure if yours is the case of DNA inhibiting PCR or that, the residual extraction buffer component(s) in the final DNA solution that's inhibiting your PCR.
Since you're working with mosquito DNA, I think the DNA yield shouldn't be too much to inhibit the PCR?
It's my presumption that, by diluting the DNA sol, you'd diluted the probable cause of inhibition to your PCR, but still maintaining the DNA quantity detectable by PCR amplification.
Thanks for the reply, I'm not convinced it's residual extraction buffer components that carry over to the PCR. The QIAamp DNA mini-kit includes a "dummy-spin" step after the last wash buffer is applied (centrifugation step with no reagent added), I also 'air-dry' the spin column for 3 minutes before adding the elution buffer.
I know I've heard the saying "too much DNA inhibits pcr" on plenty of occasions!!!!
I don't understand how though.
I would have to think about concentrations,
but if you have more DNA than primers and polymerase, you might have bad chances that you will get primer and polymerase binding on the DNA.
you need an excess of primer compared to DNA.
The amount of template depends also on the type of DNA. In animal genomic DNA (with large genomes and unsheared fragments) you have relative low DNA concentrations (i.e. parts/volume; nmol/ul), so you can use up to 1 ug per reaction (but is not recommended). If you use cDNA, plasmid DNA or bacterial genomic DNA, a sufficient amount can be in pg or ng range, the genome or the parts are small so the concentration much higher.
I did accidently a PCR with about 10 ug insect DNA (and precut with restriction enzymes) in 50 ul total volume. The problem was to identify the PCR product from template DNA on the gel, but finally we found out that it worked, it was only waste of valuable sample DNA.
To much RNA in genomic DNA samples also inhibits the PCR reaction, I always add RNase in one of the sample preparation steps. RNA binds Mg ions so that the concentration is too low later.
Does to much DNA not also bind Mg and inhibit the reaction?
P.S. For my standard PCRs with Diptera-DNA, the normal amount of DNA is between 20-200 ng (in 25-60 ul total), no problems, if the primers are ok.
Imagine the case where every extension reaction is primed on the template, rather than on the product of a previous cycle. What you get is not a clean single length PCR product, but rather a smear, with the average length controlled by the extension time and the processivity of the enzyme. To get a clean product band, you need a large excess of the product band which is then primed and amplified. With huge amounts of template, the template binding sites compete with the product binding sites, and may win.