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Old 01-29-2014, 08:12 AM   #1
flobpf
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Location: USA

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Post Method for targeted sequencing

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)...
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Old 02-03-2014, 01:21 AM   #2
riv
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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, Im interested too.
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Old 02-03-2014, 08:41 PM   #3
SNPsaurus
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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.
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