Oh that preprint suggesting SARS-CoV-2 has fingerprints because someone had fiddled with the genome.
First, you can indeed make a recombinant SARS-CoV-2 from cloned cDNA using the type IIS restriction enzyme BsaI (and another one) 👇 by Xie et al
1/21
(sciencedirect.com/science/articl…)
First, you can indeed make a recombinant SARS-CoV-2 from cloned cDNA using the type IIS restriction enzyme BsaI (and another one) 👇 by Xie et al
1/21
(sciencedirect.com/science/articl…)
Type IIS restriction enzymes like BsaI and BsmBI are useful to generate recombinant virus genomes, and Bruttel et al suggest that the distribution of the cutting sites along the SARS-CoV-2 genome is "a fingerprint indicating a synthetic origin of SARS-CoV-2"
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Some basics:
Restriction enzymes are cutting DNA, and each enzyme is specialized for a specific sequence. When you cut two different pieces of DNA with such an enzyme, you can glue them together to get a new piece of recombinant DNA.
(sharedocnow.blogspot.com/2020/01/to-cre…)
3/
Restriction enzymes are cutting DNA, and each enzyme is specialized for a specific sequence. When you cut two different pieces of DNA with such an enzyme, you can glue them together to get a new piece of recombinant DNA.
(sharedocnow.blogspot.com/2020/01/to-cre…)
3/
The advantage of type IIS restriction enzymes like BsaI is that the recognition sequence is different from the cutting site.
(neb.com/products/r3733…)
GGTCTC: BsaI recognition sequence
Arrows: cutting sites in the two DNA strands
"N"s mean that any sequence is fine to be cut
4/
(neb.com/products/r3733…)
GGTCTC: BsaI recognition sequence
Arrows: cutting sites in the two DNA strands
"N"s mean that any sequence is fine to be cut
4/
So you can design your experiment in a way that you first add artificial recognition sequences (red bars in the figure) to direct the cut to where you want it, and the recognition sequence then removes itself.
(researchgate.net/publication/27…)
Look how the red bars are eventually gone!
5
(researchgate.net/publication/27…)
Look how the red bars are eventually gone!
5
That's called seamless cloning and is used quite often to assemble big chunks of DNA, like when you need them for making recombinant coronaviruses (which have a very big genome).
This is what they did in that paper from tweet 1
6/
This is what they did in that paper from tweet 1
6/
Importantly, for each fragment you want to stitch to others, the trick is to use an enzyme that has no internal recognition site. That's why Xie et al used BsaI for the 5' (left hand) 2/3rds of the genome.
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...and in the text Xie et al. emphasize that there is no trace left of the initially added BsaI recognition sequences
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Now compare how Bruttel et al. (biorxiv.org/content/10.110…) depicted the cloning process: in their cartoon, the final product has left-over enzyme recognition sites (shown as red bars in panel G))
Hm...
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Hm...
9/
You CAN actually do it like that, but why should you? It's not necessary and actually more complicated.
However, showing the standard method instead (see Tweet 5) would have robbed them of their entire argument.
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However, showing the standard method instead (see Tweet 5) would have robbed them of their entire argument.
10/
Lets now look at what they proposed "How to make a SARS-CoV-2 BsaI/BsmBI restriction map from close relatives"
According to them, you would need to introduce at least 5 additional mutations into one of the other viruse genomes (remove-add)
11/
According to them, you would need to introduce at least 5 additional mutations into one of the other viruse genomes (remove-add)
11/
But as mentioned you can do that much easier with the standard method. Just add external, restriction sites to the primers, and use BsaI where there are internal BsmBI sites and vice versa
I'll show this for BANAL-20-247:
12/
I'll show this for BANAL-20-247:
12/
Once you figured out which enzyme works best where, design PCR primers with recognition sites for the respective other enzyme (BsaI ends for regions with internal BsmBI sites and vice versa), and also divide into smaller fragments (technical reasons):
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Then you use those two enzymes to cut the ends of the fragments (the reactions are in separate vials).
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Thereby, each overhang is compatible with just one other overhang. Now you can pool the cut fragments, and there will be exactly one possibility they can combine. Molecular Tetris
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There you go with "How to make a SARS-CoV-2 BsaI/BsmBI restriction map from BANAL-20-247"
The "Remove 4, add 1 restriction sites" as Bruttel et al propose is much more work and not required.
It is sufficient you design the right PCR primer sequences.
16/
The "Remove 4, add 1 restriction sites" as Bruttel et al propose is much more work and not required.
It is sufficient you design the right PCR primer sequences.
16/
Proposing a remove-and-add-strategy is only helpful if you want to imply that SARS-CoV-2 contains telltale footprints of genome fiddling.
You can do that without leaving any such footprints, and it even saves you time, money and work.
As Xie et al did in their paper
16/
You can do that without leaving any such footprints, and it even saves you time, money and work.
As Xie et al did in their paper
16/
Not unimportant to add that BANAL-20-247 and the other viruses have hundreds of nucleotide differences to SARS-CoV-2 that are spread all over their genome.
So you just cant make a SARS-CoV-2 out of them by some remove-and-add plus fragment cloning.
That figure is nonsense
So you just cant make a SARS-CoV-2 out of them by some remove-and-add plus fragment cloning.
That figure is nonsense
And yes, also other aspects of the preprint by Bruttel et al seem like, well, I let others speak here
/18
/18
https://twitter.com/K_G_Andersen/status/1583252858740219906?s=20&t=OTNaJ1nU8gLRdPdrb1ljRg
For this thread I ignored my overcrowded mailbox, kicked my to-do-list out of sight and may have to stay up until late to finish some really urgent work
Good night
Good night
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