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Old 01-29-2014, 07:12 AM   #1
Location: USA

Join Date: Apr 2010
Posts: 76
Post Method for targeted sequencing


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, 12:21 AM   #2
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Location: Leipzig, Germany

Join Date: Jun 2011
Posts: 3

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, 07:41 PM   #3
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Location: Eugene, OR

Join Date: May 2013
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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, or MYcroarray for larger regions.
Providing nextRAD genotyping and PacBio sequencing services.
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illumina, non-model organism, sequencing

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