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Problems with designing a primer - (Jun/10/2010 )

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.

3) Obtain file in FASTA fomat and use Genefisher2 primer design web based program ( 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!!!


Dear nesta,
Try primer3 at
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.


-adrian kohsf-

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".