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Problems with designing a primer

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#1 nesta_gwl



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Posted 10 June 2010 - 07:03 AM

Hi all,

I have a problem with designing primers. I am trying to detect a series of related viruses in tissue samples and I am now designing primers to use.

Below are my steps.

1) Obtain sequence samples from genbank. (12 sequences - whole genome ~7800nt)

2) Align them using multiple sequence alignment tool. http://multalin.toul...ra.fr/multalin/

3) Obtain file in FASTA fomat and use Genefisher2 primer design web based program (http://bibiserv.tech...de/genefisher2/) to help design the primers.

This is where the problem lies. The program cannot find a primers based on the sequences, even when I adjusted the parameters but still it failed to design a primer. It always says, 3'GC underrun, primer GV underrun, etc.

So i decided to check through the sequences manually and I have found out that the sequences have alot of conserved regions. It is just that the conserved regions (fully identical) are like 8-9bp long and separated by a few nt that are not similar, and contines to be another 5-9bp long which are conserved. and this pattern continues for another 1000bp.

So I wonder is there any better open source primer design programs to help me?

Another question is that to design a primer for these sequences. Do I have to only look for regions that are 100% conserved within these 12 sequences? So far, I cannot find any sequence that fulfills the criteria of a primer with 100% similarity. Can I just replaced the nt with the most occurring nucleotide between the 12 sequence.

Eg: ataatgc(a/g/c)gggcacttgc

Lets say I have a primer which is like the above. At the 8th nt, it varies between 3 nt within the 12 sequences. Can I just put it in such a way that since "a" occurs in 7/12 of the sequences, I will use "a" to design the primer. Will it work? If not. Is there other way??

Thank you in advance!!!

#2 Adrian K

Adrian K

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Posted 10 June 2010 - 08:16 AM

Dear nesta,
Try primer3 at http://frodo.wi.mit.edu/primer3/
use your consensus generated.

Perhaps you can design a degenerate primer. "Usually" if is only one or two bases mismatch it will not affect the binding to the DNA template.

Again, primer design is always try an error.

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#3 HomeBrew



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Posted 10 June 2010 - 12:21 PM

You can order degenerate primers. Check with your primer synthesis company, but, for example, if you're trying to match ataatgc(a/g/c)gggcacttgc", you can order "ataatgcvgggcacttgc" and get back a mixture of primers (one-third ataatgcagggcacttgc, one-third ataatgcggggcacttgc and one-third ataatgccgggcacttgc, in this case) -- "v" is the IUPAC nucleotide ambiguity code for "A or C or G".

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