DMSO increases specificity of pcr but i would try the pcr without it as it does also inhibit pcr a little. It could be that this part of the ffpe dna has a polymorphism at the 3'end of one of your primers which does not exist in your control dna  so move your primers in/out a little or drop the annealing temperature 4 or 5 degrees. If the dna is degraded in this area due to its sequence and heavily nicked you could try repairing the dna with klenow fragment and ntps prior to amplification in case your template is simply falling apart at the first denaturation stage of the pcr. If all else fails re amplify your product with nested primers in case there is some product there but too little to see

I confirm the previous suggests.You can also try to use a different enzyme or Mix.  Moreover sometimes you  might have a high GC rate in your region and you should use an appropriate enzyme. In this case i usually use the STAR GXL from Takara. It works most of the times.

Paul, when I amplify the region without DMSO, no band was seen on positive control. Therefore, I used DMSO in the reaction. Is it possible for my FFPE, I should omit DMSO? I will try to drop the annealing temperature first. Thank you Antonio for your sugguestion.

hi,

as the reaction needs dmso you must keep it in the mix but i agree dropping the annealing tempetature is worht trying and in case the template dna is gc rich you could add betaine to a final concentration of 1molar to your mix.this helps the target dna to melt easily so makes pcr work better. it is odd that the reaction needs dmso which usually cleans up dirty amplifications by making the primer stick most stringently. I think it could be affecting the melting of the template which might suggest that the template is gc rich or has a high melting point domain in it hence my suggestion of betaine in the mix. good luck

paul

Thank you Paul.

Hi Paul, I have try as you suggested, lowering annealing temperature and adding betaine. I have also try other master mix and enzyme. There is still no band on the FFPE sample. Should I change to another sample to try?

thats a shame, does your  positive control work in the current conditions as well as a negative control in your reaction which hopefully gives no band....it is always good to know that all of the reagents are working when one sample fails to work. I would also use a different primer pair if possible to another amplimer as it is possible that the whole sample is simply too degraded to use in pcr.  Look also in the online databases for polymorphisms at the primer positions particularly near the 3' end of each primer if the positive control works just in case there is a polymorphism stopping the 3' end of the primer sticking which would stop the reaction completely.

If all else fails it may be that you have some product but not enough so you could try nested pcr where you design primers just inside your primers and run a few cycles of pcr on very dilute product from the first reaction....the primers do not have to ve very good generally as there should be plenty of targer in the second reaction I just move the primers inabout 10 bases from the 5' end of the original primers making sure that the 3' end of each primer is inside the positions of the original primers

good luck

Hi Paul,

My negative control has no band while my positive gave bright and several non-specific bands. I am taking up your suggestion regarding nested PCR. Lets see what will happen then. Thank you.

i would be interested to know how it turns our Lee Chai. Good luck. Paul

Ok Paul.