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closeThe cfDNA is present as nucleosomal particles
Posted by alakdawalla on 26 Aug 2017 at 22:58 GMT
Nice paper. Have a question on whether the thermocycling conditions release the DNA from the histones so that it can be amplified? Would using a thermolabile protease pre-treatment show even higher cfDNA levels?
Curious.
RE: The cfDNA is present as nucleosomal particles
simonpe replied to alakdawalla on 27 Aug 2017 at 14:44 GMT
We have compared our direct measurements with the measurments of cfDNA isolated by using very old and very danaturating conditions. While most silica-based isolation protocols seem to underestimate the problem of the nucleosome as a highly stable DNA-protein-complex with the DNA protecting the protein from proteinases and vice versa, the old methods revel roughly the same concentrations as our direct amplification technique. One critical point is to ask companys selling their DNA-purification product for data on retrival ratios following chromatin and not following spike in with naked genomic DNA.
Why is our procedure detecting nucleosomal particles?
Most likely at >90°C the double stranded DNA is releasing at least one strand from the nucleosomal particle, which may serve as a starting point for subsequent exponential DNA amplification. At >70°C it is still highly likely that affinity of most polymerases for DNA is high enough to even amplify the strand still loosely attached to the histone complex.
Taken together, I am very convinced that our direct detection is not only cheaper and faster, it is also helping to circumvent some problems with technical and biological issues causing variation in DNA purification. Please note: One of the buffers we used had been removed from the market. We will publish a protocol soon to circumvent this problem.