Hi,
I don't have much of a genetics background and wanted to ask for some advice. I was looking to produce primers for a gene encoding a certain enzyme, MC5E, found in brown seaweeds. There is an annotated sequence available (introns and exons) for the gene for Saccharina latissima and a genome assembly for Ectocarpus siliculosus on NCBI GenBank.
When I blasted the assembled genome for the individual exons I got hits on a bunch of different chromosomes with reasonably high % cover and query identity, making me think that some amount of trans-splicing is occuring (or potential polyploidy is confounding this). I then went to see if there were any chromosomes over represented in the blast, and came up with 4 Linkage groups/chromosomes on the assembled Ectocarpus genome which generally showed good hits for my exons. After blasting the Ectocarpus genome for the gene CDS, I got a bunch of hits with minimal cover but decent %, and when I visualised these as a "Distance of Trees" I found those same Linkage groups clustering.
My question is, how do I best approach my primer design in order to be able to catch SNPs likely to influence enzymatic functioning? Originally I intended on just capturing the whole gene, but now it looks like I may need to design primers for the exons specifically because it appears to be spliced from across genome regions. The goal is to be able to look at the variation in this gene between species and populations, with some nod to functionality by looking at its AA translation.
I hope this makes sense, and please let me know if there is anything I missed.
Simon
My first thought is that the MC5E gene may be part of a larger family of enzymes with reasonable similarity. I know next to nothing about the taxa you mentioned, so this is not a rhetorical question: is trans-splicing really more likely than the presence of multiple genes from the same family?