Hi everyone,
I'm looking for a mean to specifically identify a single stranded DNA in a gDNA prep where double-stranded DNA is largely predominant. This ssDNA, I want to detect is derived from dsDNA cleavage I generate.
I tried to detect it by PCR following TdT reaction. I carried out TdT reaction to add polyC tail directly on my gDNA prep with the hope to target 3' end of the ssDNA I want to detect. And then I performed PCR using a primer specific to the single stranded DNA and an other specific to the polyC tail.
I detect a signal but my problem is that I detect the signal even in a condition where the cleavage should not take place (and where the ssDNA sould not be present). I think, with this method I also amplify ssDNA generated by normal cell metabolism.
Do you know a mean to detect this ssDNA without the background noise I detect ?
Could you anneal it to an immobilised antisense oligo ( on bead/nylon/streptavidin bead and biotin oligo)then wash away all dsDNA and non specific ssDNA to purify the target dna before running the pcr ?
Hi, thank you for your answer !
I've already tried to amplify the targeted ssDNA with a biotinylated forward primer and a reverse primer specific to the polyC tail. Then, I captured biotinylated PCR products with Streptavidin beads. I thought it would solve the problem but it didn't : I still detect a signal in a condition where the cleavage should not take place... I think I amplify with this method Okazaki fragments (for example) which could have a free 3' end (targeted by TdT) and a region specific to my biotinylated primer...
Hi Leo,
I agree that using the same oligo that produces a poor result in pcr is not a good idea. I was thinking of using a more specific primer to pull ot the template from the mixture. So if your template can be represented by bases 1-100 then pull out the template with oligo antisense to 50-70 then wash away the unbound material and pcr with 1-20f and 100-80r. I was hoping that the non specificity of you pcr was generated by the end primers that you used and that maybe a different sequence in some part of the process might help
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