Primer Schemes for Divergent Viruses

[AMPByrne]

Hi,

More of a question than an issue, but in the README it suggests that is you have greater than 5% identity divergence between the sequences you are using to design a primer scheme, that you may have to split these out into separate schemes.

I'm trying to generate primer schemes for some veterinary viruses, where there is a lot of sequence divergence within viral species. I'm happy to split them out into separate identity groups and then design separate primer schemes for each group, but I was wondering, can I then just pool the primer sets for each one and run as a single RT-PCR reaction? Would this work or would there be too much primer-primer interference?

Thanks in advance for you help!

[joshquick]

You can't mix them and guarantee that you won't get primer dimers formed because there's no checking across schemes when you run them separately. I would run the PCRs separately or manually confirm lack of dimer products when you mix them.

We have used the primers for cDNA generation before but when I last tested it I found it wasn't as good as random priming. That may not be true now and I believe NEB have a new version of the ARTIC kit which does actually do that.

Josh