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Primers design problem

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Primers design problem

Postby magicsiew » Fri Dec 17, 2010 9:59 pm

Hi, another post on primers design. I have to design primers to amplify a gene, but then has no annotation of that particular gene in Genbank. So what I do is find the gene sequence from related species-found 6 protein sequences and have only these 6 available (even less in nucleotide sequences). When I aligned them, it shows one conserved region only, very obvious one only. So, I can use that region as either forward or reverse primer. My problem is how to solve this problem, since we need both forward and reverse primer. Besides this conserved region, the other (the most a.a conserved) region is 4a.a only, which equal 4x3=12 nucleotide only, definitely not enough long. Anyone has any idea on how to solve this problem? Thank you.
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Postby Kezzer » Sun Dec 19, 2010 12:08 pm

Hey am I right in thinking that you've been given an amino acid sequence and asked how you would amplify the gene from that sequence? If so make sure if you're using related sequences that they are identical to the gene you want to amplify, there is usually a link on NCBI blast to tell you this.

If you only have the amino acid sequence (no nucleotide data) to amplify the gene obviously use the most conserved regions to design degenerate primers from, so use the one conserved region to design your first primer....you can use another region to design a primer even if it's not that conserved as highly degenerate primers can still work up until a certain point.

What methodology were you thinking of using? With degenerate primers you can use RT-PCR and then design specific primers for the product to do PCR to obtain the gene of interest.

If not have you just been given the name of a gene to amplify??
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Postby magicsiew » Sun Dec 19, 2010 1:22 pm

Yes, I am been given a name of a gene to amplify. I have done PCR/RT-PCR as what you suggested and the sequencing results come back all shows negative results. With only 2 results when BLAST-ed, shows my gene of interest, however is too low the percentage of coverage (2% only).

Then I try to identify that 2 regions in BAC Clones complete sequence (only BAC CLONE available in GenBank), copy regions around and BLAST with it, all dint not show any results of my gene of interest.
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Postby Kezzer » Tue Dec 21, 2010 9:00 am

Well I don't know what to say other than try again I'm afraid.
Sorry I can't be more help!
Hope you get it sorted :)
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Re:

Postby JackBean » Tue Dec 21, 2010 10:10 am

Kezzer wrote:Well I don't know what to say other than try again I'm afraid.
Sorry I can't be more help!
Hope you get it sorted :)


what a great answer :lol: :roll:
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Re: Re:

Postby Kezzer » Tue Dec 21, 2010 1:14 pm

JackBean wrote:
Kezzer wrote:Well I don't know what to say other than try again I'm afraid.
Sorry I can't be more help!
Hope you get it sorted :)


what a great answer :lol: :roll:


Well I said what I knew in the first post.......better to say you can't help further than to tell someone the wrong answers in my opinion :P You offering up some help with this topic? :lol:
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