Primer Designing

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LAKSH
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Primer Designing

Post by LAKSH » Mon Apr 09, 2007 11:53 am

Hi, I am working on a big gene in Drosophila and I have to find out its presence in a specific tissue of the fly from the tissue specific mRNA.
I have questions regarding designing the primers for the gene:
1. I am planning to use the coding region of the gene which is approximately 6kb size for the primer designing. Is this fine or should I select the mRNA sequence of the gene to design the primers?
2. When I am designing the primer for this gene sequence should I feed the whole sequence (mRNA/coding sequence of the gene) in the primer designing program Or should I select a part of the sequence. If so, what are the criteria in selecting the sequence region to design the primer, to get promising results?
3. Can the primers be designed at the 3' or 5' UTR of the mRNA if they have low GC content?
Thanks in Advance for the great help.
Cheers,
Laksh

blcr11
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Post by blcr11 » Mon Apr 09, 2007 12:34 pm

You are designing just cloning primers? So long as you know the sequences for 30-40 bases downstream from the start and 30-40 bases upstream from the stop codons, you can design the cloning primers by hand. Just look to see if you have any undesired potential loop structures and scan the rest of the coding sequence to see if there might be a problem with internal sequences that might give false-priming artefacts; you can either modify the primers a little or modify the pcr protocol if you know there's a potential problem there. If you're also designing sequencing primers at the same time, you need to feed in the mRNA sequence. If you feed in the genomic sequence you'll probably design some primers that hybridize to introns and those primers won't be useful--I'm assuming that the coding sequence is in fact the desired target.

You can design primers to capture 5' or 3' untranslated regions if you want to. Most of the time you don't want to. Is there something of potential interest there?

LAKSH
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Post by LAKSH » Fri Apr 13, 2007 9:54 pm

Hi blcr 11,
Thanks for the reply.
Can you please tell me how to find the undesirable region and other mis priming sites of a gene sequence when designing the primers. Is there any special programs available for this?
I am designing primers for sequencing and hence I am using mRNA. The reason for asking if 3'UTR of a mRNA could be useful in designing the primer is: I thought that since this region preceeds the poly A tail, we can easily get the gene amplified from the cDNA reverse transcribed using oligo dT primers.
Would be glad to know your thoughts on this!
Laksh

blcr11
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Post by blcr11 » Sun Apr 15, 2007 8:57 pm

I usually design the cloning primers by hand. You can check oligos for self-loop-stem structures at http://www.basic.northwestern.edu/bioto ... ocalc.html As for checking for potential false priming, there isn’t any terribly good program I know of. I usually Blast the oligos against the target sequence at low stringency and look to see if there are any significant “homologies.” You can design sequencing oligos by hand if you want; I use a program called Oligo http://www.oligo.net/ but it’s not free. One program available for free that looks useful is PerlPrimer http://perlprimer.sourceforge.net/. You might also try the webservers GeneFisher http://bibiserv.techfak.uni-bielefeld.de/genefisher/ or MEDUSA http://www.cgr.ki.se/cgr/MEDUSA/. I can’t really speak for any of them since I’ve never really used them except for the oligonucleotide properties calculator, which works just fine.

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Post by blcr11 » Tue Apr 17, 2007 6:14 pm

I was looking for something else when I stumbled on this page that has a number of tools, including a primer design tool. The company makes it freely available.

http://www.genscript.com/gene_tools.html

SororSaudade
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Post by SororSaudade » Wed Apr 18, 2007 12:46 pm


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