I'm looking for a bacterial DNA in fish genomic DNA. Primers have proven their efficiency by bacterial DNA directly but working with tissue, It results in so many non-specific bands. I have tried temperature optimisation or working with different template concentration (not sure the higher amount of DNA would help or the lower). I even changed the amount of primers I used, time of initial denaturation and final extension. None of them helped. I doubted maybe the fact that I kept tissue samples in 96% Ethanol before DNA extraction caused a problem so I homogenised fresh tissues and again extracted but again no difference was observed.
Any idea regarding what else can be done to detect the bacteria DNA in tissue is more than appreciated.
P.S: Is it possible that bacterial circular DNA integrates fish genomic DNA after entering the tissue?
Apologies for the long explanation,
Many thanks in advance.
Dr. Negar,
This is quite a common problem while working with samples where host DNA is in abundance with respect to the target pathogen DNA. I would suggest you to do a RE digestion of an aliquot of the sample DNA extract with an enzyme that doesn not cut within your target amplicon region. This will help to fragment the host DNA, rendering them lesser effective as PCR templates, while linearization of the supercoiled bacterial DNA by RE digestion will help in increasing the sensitivity and specificity of your PCR. The selection of the RE to be used is important and should be done in a way that it has no site within your target amplicon, but has frequent cutting sites within the host DNA or other parts of the bacterial DNA. for your reference you may read the follwoing paper:
https://www.researchgate.net/publication/307559700_Datta-Mol_Diagn_Ther-2016
best wishes,
SD
Data Datta-Mol Diagn Ther-2016
the possibility of integration of bacterial DNA within the fish genome seems to be very rare, if not completely impossible. But the relative abundance of fish DNA vs. pathogen DNA seems to be the possible reason for this.
is your primer for 16srRNA , if that may be there are more than one bacteria species which cause that, if not you can use more specific primer by check on NCBI which not work on fish genomic
Dear Dr. Datta,
Your time and valuable points are highly appreciated. Many thanks for introducing me your article as a great reference. Guess now I should focus on finding an appropriate RE to cut both fish and bacteria DNA so that the target region could be more easily accessible.
Myriads of thanks again,
All the best.
Dear Dr. Suleiman,
Many thanks for your time and support. I am using several primers and this problem exists in all of them.
Shokran jazila:)
Best regards.
For the PS part, no it's not a possibility. Even if it was, that would not be a reason your PCR isn't working properly. Are you sure you are seeing "non specific bands" as opposed to DNA from several bacteria species? My best guess is that either your annealing temperature is not correct or you are using too many amplification cycles. More than 30 cycles increases your chances of artifacts.
What method are you using to extract DNA? You might want to try a DNA extraction protocol that gives a very clean, very pure DNA product. PCR is sensitive enough that less DNA is almost always better than more DNA. Try a dilution of your DNA (full strength, 1:10, 1:100).
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