Method for targeted sequencing

[flobpf]

Hi,

My goal is to obtain the sequence of a gene from several species. I know the sequence only in 2 species separated by ~30mya. Designing optimal primers is not an option, since if the PCR reaction doesn't work in one species, I wouldn't know whether its because the primers failed or the gene is absent. Southern is too cumbersome, in my opinion. Wonder if there is another way to do my job? I checked for commercial kits but most kits for targeted sequencing are in humans and I'm working in a non-model plant lineage.

I want to stress that I only want to get only selected loci (5 genes in my case)...

[riv]

Hi! I am not sure if I got your question right, but I have these ideas:

These genes that you are interested on maybe have some region that is more conserved and therefore it can be amplified with PCR. Even if you get a tiny part, you can do RACE PCR after that.

If you only have the sequences in 2 species and you want to check which parts should be more conserved, you can look for those genes in another taxa and make some multiple alignment.

Anyway, I know that optimizing PCR can be really annoying. If somebody has another idea, I´m interested too.

[SNPsaurus]

For isolating DNA that has high divergence, pulldown by hybridization is usually better. You can order pulldown baits from any oligo supplier, such as IDT https://www.idtdna.com/pages/product...target-capture, or MYcroarray for larger regions.