mismatches in primers

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Gerald Weber

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Apr 15, 2020, 4:36:09 PM4/15/20
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Dear All

I've only a passing understanding of LAMP technique coming from a few papers I just read these days, and not sure of what the primer requirements are. So to better understand the primer design I played around with aligning the primers to some random virus genomes. 

While doing this, I noticed that the C-F3 would hybridise with one AC mismatch in MT106054.1 2019-nCoV/USA-TX1/2020 (which was picked at random)

CTGCCAAAAGCTTGTGTT (complementary genome 5'->3')
GACGGCTTTCGAACACAA (primer 3'->5')

a rough calculation suggest a 4 C reduction in Tm

I have not tried other SARS-Cov-2 genomes, so I don't know if there are more cases like this one.

So my question is: how relevant or problematic is this? Does it even make sense?

The other question is, would it make sense to repeat this for other SARS-Cov-2 genomes and evaluate the reduction in Tm? 

My group has just finished a major thermodynamic analysis of all combinations of mismatches in DNA (https://doi.org/10.26434/chemrxiv.11977887.v1) , some of which can be surprisingly stable. It should be possible for us to do this.

best regards

Gerald


Dineshkumar Dandekar

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Apr 15, 2020, 9:31:43 PM4/15/20
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Thanks Gerald
for your insights and observations.

Please have a look on list of primers posted on Primers of RT-LAMP thread.

Let me know your thoughts and comments

Regards
Dinesh

Mikolaj Slabicki

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Apr 15, 2020, 10:57:11 PM4/15/20
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Hi Gerald,

 

In the pre-print publication, we analyzed the alignment of our primers against 183 SARS-CoV-2 genomes. Every primer set perfectly matched >95% of all viral genomes. Please have a look at the supplementary file attached to the manuscript.

 

I think a single mismatch would inhibit the LAMP reaction but this would have to be tested experimentally.

Potentially one should use more than one set of the primers to cover all SARS-CoV-2 variants.

 

Thanks a lot for your comment.

 

Cheers,

Mikolaj

Gerald Weber

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Apr 16, 2020, 8:37:18 AM4/16/20
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Dear Dinesh

your primer proposal aligned completely with that particular MT106054.1 genome, but as said, that was just one randomly picked. We will now to the same for the complete set of genomes and see how it goes.

best wishes
Gerald

Gerald Weber

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Apr 16, 2020, 8:45:14 AM4/16/20
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Dear Mikolaj


In the pre-print publication, we analyzed the alignment of our primers against 183 SARS-CoV-2 genomes. Every primer set perfectly matched >95% of all viral genomes. Please have a look at the supplementary file attached to the manuscript.


thank you very much for point this out. Indeed, I just feel so stupid now as I was staring at your primers and not realising the additional sheets in your excel file.
 

I think a single mismatch would inhibit the LAMP reaction but this would have to be tested experimentally.

Potentially one should use more than one set of the primers to cover all SARS-CoV-2 variants.


using more than one set of primers makes a lot of sense. 

Still, we can just check out the Tm's for the non-matched primers and see how it goes. Some 15% of single mismatch context have higher Tm's than the least stable AT base pair, so interesting things could happen. We are in the process of setting up our scripts for this, I'll post the results here when we are done. Hope this may be of some use.

best wishes

Gerald
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