Hi there,
I would like to do a tumor burden follow-up by measuring circulating cell-free DNA (ccfDNA) at different time points in a mouse experiment (by treating or not mice after tumor initiation).
Does someone have a protocol to amplify DNA as I can draw only 100-200µl of blood/week in mice.
I have a protocol to isolate DNA (QIAmp DNA blood mini kit) from plasma.
Thanks in advance!
Christophe
Dear Christophe,
I do not have a protocol as such but if you can get hold of Droplet PCR (by Biorad), you should definitely give it a try. I attended a talk recently that addressed the exact same issue as yours with the Droplet PCR.
the amount of circulating dna will be small compare with the parental dna so I would expect that you will need nested pcr with 2 pairs of primers to preferentially amplify your target
Dear Paul, are suggesting to put 2 pairs of primers instead of only 1 pair to amplify DNA?
Thank you for your responses!
Bests
Chrisotphe
i am suggesting a purification of small dna with somethinh like the Qiagen viral dna kit then you will have very little dna target of interest compared with the parental normal dna so a first round of pcr with primers identifying your target of interest followed by a re-amplification of a high dilution of that pcr product with primers internal to the first set but specific for the dna that you are interested in...possibly ARMS primers if you know that the circulating tumour dna has a polymorphism at a useful point so that this time you only amplify the tumour dna. This is not necessary if you already have a gene marker that only occurs in the tumour then you can just rely on the first round amplification
Thanks a lot Paul, I'll come back to you when I would have time to retry it!
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