Original with graphs at above link:
Additional follow up on DNA contamination of COVID-19 injectable products (there's more coming too)
There are circular plasmids (DNAs) as contaminants...\
The saga of work being done in the lab context to confirm or deny the presence of DNA contamination in the Pfizer and Moderna shots continues. Unlike ‘bored’ meetings held behind closed doors, lab procedures take time and care (also behind closed doors, but for other reasons), and no matter how much you might want to, you can’t hurry love. Or, lab results. ‘Tis better to be smart and cautious, than first.
…The results continue to roll in like a glassy 1 meter wave at day break in a place that no one knows about.
Anandamide’s newest piece is entitled: “Sequencing of RNase A treated Pfizer bivalent vaccines reveals paired-end sequencing evidence of circular plasmids and an inter-vial 72bp variation in the SV40 promoter” and it confirms the presence of circular DNAs, as opposed to linear DNAs, as contaminants in the tested vials. By the way, if you want to watch an amazing documentary about the origins of the name Anandamide, please do. It’s called The Scientist, and it’s very relevant to the crushing blows that real science sustains, to this day.
To summarize the current situation, Kevin’s team sequenced both the RNA and DNA in samples of Moderna and Pfizer injectable products. They used an RNA-Seq kit but since this method favors RNA sequencing, the realized it might actually underestimate DNA contamination levels. Since they wanted to assess the amount of DNA contamination and quantify the ratio of linear to circular plasmid DNA, they went one step further and RNase A-treated the samples (this gets rid of any RNA in the samples) and sequenced the remaining DNA.
By the way, whenever you see ‘-ase’ tagged onto a word, it means that you’re dealing with an enzyme which is simply a biological catalyst of a chemical reaction. So RNase just acts to break up RNA into little RNA bits. Kind of like when you cook spaghetti too long and it breaks up into smaller bits when you take it out of the pot. Sort of. The real question is: is it still spaghetti?
Overcooked spaghetti wand Base Damage:1 Durability 0.5 When Equipped:Immediatly breaks unless ...
The following screenshot is from this latest article and shows the Pfizer version of the plasmid vector on the left and their RNase A sequencing and assembly-produced vector on the right.
The first thing I noticed was that the Pfizer disclosed plasmid contains the poly-A tail in the plasmid sequence. As a point of interest, this saves the step of having to add it after mRNA production however, it is problematic because bacteria don't support long stretches of As (anything longer than 80 AAs is too many). Longer poly-As can cause recombination events to occur which is bad for clinical material production. Also, if you wanted to extend the poly-A tail, you can use a poly-A polymerase afterwards, but this costs a lot of money to do. It could also cause problems in terms of the different distributions of poly-A tail lengths that would arise which, of course, would have to be measured and monitored, ie: reproducibility of poly-A tail lengths between batches using qualitative (and quantitative) methods. We all know how good the quality control is with regard to these products, so if they did add poly-As postfactum, I am sure they maintained consistency across the board at all the different manufacturing sites. Sarcasm detector ‘sploding. Here’s the screenshot.
Here are some definitions that I probably should have already provided:
In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter.¹ It controls gene expression.
SV40 is an abbreviation for Simian Virus 40, a polyomavirus that is found in both monkeys and humans. It is a mammalian promoter with high expression driving ability with close affinity with RNA polymerase and can direct the massive synthesizing of mRNA. It improves the gene expression level of many host cells.² It promotes expression of particular genes in mammals.
The origin of replication (ORI or ori) is a particular sequence in a genome at which replication is initiated.³ It's where replication starts.
KanR is a kanamycin resistance gene cassette.⁴ It confers kanamycin resistance.
Back to the nitty gritty.
The orientation of the paired end reads (they point outward - seen in green) also provides strong evidence that the plasmid is circular. Referring to the original EMA document entitled: “Rapporteur Rolling Review critical assessment report - Quality aspects” dated November 19, 2020, where Pfizer were told that they had to address some serious problems, on page 32 of the downloadable document provided by Sasha, it clearly states the following:
No descriptions of the analytical methods used for the control of the linear DNA template nor evidence regarding their qualification/validation have been provided (OC).
This is just one of the many things that were not provided, by the way. Here’s the document. Please read it. I added page numbers.
Dear RM Agents and Readers,
Go to the link at the very top of page, I've yet to read it, need sleep. Time change, 5 to 6 inches of unexpected snow, slept in,
helping friends and family, you know:)
Looks like excellent information and was put forth by: