Primers design without annotation.

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magicsiew
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Primers design without annotation.

Post by magicsiew » Fri Aug 20, 2010 8:14 am

Hi, I am about to design a primers to target a gene that is without annotation in the GenBank. What methods should I take besides using related model plant as a reference? By the way, the gene of my target is a from a plant.

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JackBean
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Post by JackBean » Fri Aug 20, 2010 9:28 am

do you have the sequence of either DNA/mRNA or protein?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Re: Primers design without annotation.

Post by magicsiew » Fri Aug 20, 2010 9:35 am

No sequence at all for the gene I need to target. So far, what come up in my mind is using Arabidopsis as reference only...

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Post by JackBean » Fri Aug 20, 2010 9:36 am

is that gene known in any other organism?
http://www.biolib.cz/en/main/

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Re: Primers design without annotation.

Post by magicsiew » Fri Aug 20, 2010 9:41 am

Yes, so far i can find is Arabidopsis, rice, tobacco and moss (nearer related), got bacteria also, but i dont think is suitable to be used.

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JackBean
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Post by JackBean » Fri Aug 20, 2010 10:34 am

well, then just align these and see the most conserved regions. For these design primers, clone into vector, sequence and design primers pointing out ;)
http://www.biolib.cz/en/main/

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Post by magicsiew » Fri Aug 20, 2010 10:45 am

Thats what I do to find the most conserved region, but what happened is the region between the forward prime and reverse primer is too far away, and is not is different when compared among the 4 organism stated. The forward primer can be found (hopefully), but the reverse primer I worry cant work.

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JackBean
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Post by JackBean » Fri Aug 20, 2010 10:53 am

if the region between is not conserved doesn't matter, the polymerase will make the strand no matter what is there, just the primers must be OK. You can always use degenerate primers ;) Or nested PCR with degenerated primers and then with some more specific...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Post by magicsiew » Fri Aug 20, 2010 11:51 am

I am not worry about the region between. What I am worried is the polymerase able to function well or not if the length to polymerase is too long? Because the region between forward and reverse primers is very long. By the way, is there any software can design degenerate primers?

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Post by JackBean » Fri Aug 20, 2010 1:42 pm

how long is it? Usuall Pol should be able to do at least 2 kbp and there Pols for extra long pieces like 10 kbp
http://www.biolib.cz/en/main/

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Post by magicsiew » Fri Aug 20, 2010 2:02 pm

Ya, it is between the range. I think I get what you meant, thanks ^^

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Post by magicsiew » Sat Aug 21, 2010 7:42 am

Hi JackBean, I have read through the methods u suggested and understand wat i have to do to get the primers using example from the web. However, when applying to my case, I found 1 related species protein sequence only, along with another 1 partial sequence. Other sequence all on bacteria, while I am trying to design the primers to target a plant's gene. Do i have to try and error on the bacteria sequence? Or there are other methods?

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