A few followers have asked for additional data confirming our estimations of the DNA contamination because ‘This is a pretty serious claim’. We agree. It is hard to believe it is this bad as natural transcription rates with T7 would never be this stunted. It certainly made us pause and perform multiple check on this. To summarize, we have
So is this a way to fight them? Based on the product not being as specified as per the contract due to excessive contamination?
I mean if it is easier to prove the product is defective, rather than dangerous. Dangerous requires a lot more evidence, while product purity is to a set level of compliance.
This is awesome work. What a trainwreck.
Is it possible to transfect human cells with the product and culture them, then check if their descendants conserve the ability to produce spike, or have the plasmid DNA integrated into their genome?
Solid work Kevin. If you've managed to show successful digestion of vector DNA with DNAase in these samples it begs the question why their DNAase step failed (or did they even do it?)
Do you have any plans to search for specific known peptides within the samples? This contamination is being touted as no big deal and may even help as an adjuvant. Seems like crazy talk.
I have been following this trail since February- the obvious question most of us is - how do we fix people now that have been infected with antibiotics resistant plasmids ?
Thank you very much for sharing your results. One comment:
I have some trouble with this statement "A 2CT gain when amplifying RNA+DNA amounts to 4X more RNA than DNA for Spike. This is in line with the 20-35% estimate from the Agilent gels." Even though it is true that with the addition of RNA the spike signal moves to a CT 22, the vector signal, which should not be affected by the RNA, moves to a CT 28. Hence figure 2 and figure 3 cannot be compared directly in a quantitative manner.
As you said, "This is an important control as it implies the Vector assay is not performing as well in multiplexed RT-PCR as it is in multiplexed qPCR (CT =28 vs CT = 24)."
Where do they get the DNA and is it possible the NIH gave them Henrietta Lacks DNA?? WHICH WOULD EXPLAIN ALL THE CANCER! This would be mind blowing! Assuming everyone knows the story of Henrietta Lacks! Francis Collins was in charge of her samples and protecting it's use by having a Committee that included two Lacks relatives approve or reject every single lab or project who requested to use her DNA!!
Julie in Chico
Well done! Even more evidence of DNA in these vaccines!
A couple of your results here are quite interesting and maybe worth thinking about in more detail:
A) There is less shift for the spike DNA (5 Ct) vs vector DNA (10 Ct) following DNAse I treatment in the qPCR. It could be that modRNA is binding to the DNA protecting it from DNAse digest.
B) This is also seen with the RT-qPCR, but curiously the DNAse/Vector/RT-qPCR has a lower Ct than the DNAse/Vector/qPCR; despite the Vector/no DNAse/RT-qPCR Ct having *higher* Ct than the Vector/no DNAse/qPCR.
C) You have a very funky RT-qPCR amplification curve for the spike probe, which looks different to the qPCR curve using the same probe.
Elsewhere, you've pointed out the m1Ψ is a very sticky base. Putting these together, I wonder whether the modRNA is base-pairing with the DNA and interfering with both the DNAse I digest and the qPCR.
This could explain how µg of DNA ended up in the vials despite the DNAse and residual DNA qPCR steps.
Expanding on this in more detail:
1) The modRNA wouldn't just be able to displace one of the DNA strands in dsRNA unless the dsDNA is melted or denatured.
2) T7 polymerase obviously melts the dsDNA when transcribing the RNA. Could the modRNA remain bound to the DNA following transcription and/or could modRNA gain access to the template strand during transcription?
3) When the concentration of modRNA increases at the end of manufacturer, presumably the binding kinetics begin to favour modRNA rebinding to the template DNA as long as the template strand is accessible.
4) modRNA:DNA duplexes also could be present at high number in the drug substance as T7 transcription has just finished, and renaturation of dsDNA might take time especially if modRNA interferes with renaturation.
5) In addition, if m1Ψ is very sticky and elevates the Tm of dsRNA containing m1Ψ, it wouldn't be surprising if m1Ψ-RNA:DNA duplexes also have higher Tm than DNA:DNA duplexes. This may favour the modRNA:DNA remaining persistent in the sample. Not sure if there is any data on m1Ψ-RNA:DNA duplexes? I had a quick look but couldn't find any.
6) DNAse I activty of RNA:DNA hybrids is <1-2% of dsDNA (https://www.thermofisher.com/nz/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/dnase-i-demystified.html) - I should look for an actual paper reference for this. Presumably similar for modRNA:DNA duplexes.
7) This might explain why your DNAse I had a larger change in Ct for the vector probe (no modRNA base-pairing possible) vs spike probe (modRNA:DNA duplex present); A above. This could also explain poor performance of DNAse in the manufacture, as you have eluded in another comment.
8) If the Tm of a modRNA:DNA hybrid is increased - might these not melt efficiently at 95°C in the qPCR denaturing step? This could interfere with qPCR detection of DNA when hybrids are present, i.e. cause a failure of residual DNA detection during manufacture.
7 and 8 combined might be sufficient to cause large amounts of DNA to remain in the sample and be undetected.
9) Arguing against 8, in your data, the DNA/qPCR detection appears to be working well. However, you have taken the samples through purification steps that might cause the modRNA:DNA duplexes to fall apart. Presumably these are hard to form if the DNA is fully zipped up.
10) Further, Pfizer's assay for residual DNA (as described in the EMA document) is right at the T7 promoter/Kozak sequence. This might be a hotbed of modRNA:DNA duplexes because (a) the T7 polymerase can continuously melt the dsDNA giving modRNA access to the template strand and (b) there many be many more truncated RNA strands vs full length strands due to difficulty transcribing with m1Ψ.
11) If qPCR detection of residual DNA is being affected by modRNA:DNA duplexes in the sample, this presumably will *not* effect spike in of DNA used during validation of the assay. That is, spiked in DNA will probably still be detected just fine if the issue is modRNA:DNA duplexes not melting at 95°C.
12) Something weird might be going on with the modRNA:DNA duplexes explaining the unexpected result point out in B above (or it could just be a random PCR issue).
13) The funky RT-qPCR amplification curves (C above) could perhaps be related to some weird binding effects of modRNA following renaturation (or it could just be a random PCR issue).
I wonder whether one way to look for the presence of modRNA:DNA duplexes is a some kind of melting curve experiment with a RNA or DNA dye - perhaps SYBR Green I or II. It could perhaps be done on the qPCR machine. Probably need to think about this a bit more.
I would be interested to know if you think I might be on to something here, or if I'm barking up a wrong tree. Happy to expand on this in a Substack if there's thought it might be a useful hypothesis.
A lot of scientists are saying there can't be (significant) mRNA due to the lack of phosphorus and nitrogen. Can you explain why they are drawing this conclusion?
So there are as much amount of DNA in the jab as the mRNA? Did you do those sequencing test on the actual Pfizer/Moderna mRNA vials? Are those DNA found to be naked-strands and fragments free flowing in the liquid, or are they encapsulated inside the lipid nanoparticles? What is the diameter of those lipid nanoparticles as observed?
So the take-home lesson is, if you're gonna use RT-qPCR to detect spike protein mRNA from the vaxxes in tissue samples from vaxxed peeples, you are gonna want to DNAse the sh*t out of those RNA preps prior to any RT-qPCR ?
And.......extrapolating all this still further........those proposals by Leana Wen and all the other public health establishment icons to monitor the status of covid-19 infections in the population at large, via the presence of spike protein mRNA in sewage and airplane toilets, are gonna have to start pondering what happens if there are bunches and bunches of dsDNA from The Plasmid in the Vaxx in those same sewage and toilet samples...........?!
Maybe I need to take the money I was going to deposit into the SVB, and invest it DNAse manufacturers.............or in companies that make RT-qPCR kits that target genes other than spike........?!