The use of universal primer to amplify a wide range of prey from predator gut content may amplify also the predator DNA. Thus, predator-specific primer exclusion or predator-specific elongation arrest may be used. However, the common polymerase used during PCR have exonuclease activity, which can suppress the predator inhibitor process.
I wanted to know if inhibitory process may work although I used polymerase with exonuclease activity.
I would suggest using inhibitor primers that are not digestable by the polymerase' exonuclease activites, e.g.,; 3' and/or 5' blocked, thio-phospahtes, PNAs etc.. the list is long. This is a very obvious answer, maybe your problem is more subtle ?
Regards, Pascal
5' nucléase activity of taq polymerase can be blocked by adding MeO-Cl-acridine molcule in 5'end.
https://secure.eurogentec.com/product/research-acridine.html
for elongation inhibition you can try 3'end phosphate addition. Good luck.
Regards
Nathalie
Pascal, my aim is to study the diet of a fish using faecal DNA analysis. For that I have already chosen a generalist primer set that work very well on the potential prey, but that amplify also a little the DNA of the fish. Therefore, I want to inhibit the amplifcation of the fish (predator) that should be found in faeces without changing my PCR protocol or changing the reagents (Taq and primers).
In the litterature, the most use blocking primer in my topics are: modified oligos including 3'-Spacer C3 CPG or PNA. However, 3'end phosphate addition was not so much use. So I plan to use 3'spacer blocking primer or PNA.
Futhermore, I am surprising when you say Pascal that exonuclease activity do no work on PNA. Is that what you mean? As the oligos with 3'spacer is also insensitive to the exonuclease activity of the polymerase?
It could be a good news because it would avoid me to add inhibitor of exonuclease as Nathalie proposedf. I'll try to minimize the reagents to add.
Thanks for your help,
Emmanuel
If my memory is good (no warranty!)., PNAs should be up to the job. They are also likely to provide more radical blocking than end modified oligos due to higher Tm. BTW, if my reading is good (no warranty either!), Nathalie is suggesting modified oligos also, not to add additional reagents.
Pascal
Ok Nathalie,acridine is a product to add in the primer solution in amount of the PCR! Is that?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology