3' end of forward primer mismatch - (Sep/20/2004 )
I am facing a problem.. I recently started with a project where I got to amplify gene of about 600 bp. Some other fellow was working on this before and he already had forward and reverse primers...
I was trying to amplify the gene using PCR. However, each time I received a smear.. I tried several conditions.. but it wont work...
When I analysed the primers, I found that there is a 5 base mismatch at the 3' end of the forward primer.. Do you think it's a major problem? Should I redesign the primer?
I would highly appreciate any help!
Is the mismatch at the very 3' end (the last nt)? If yes, you may have to redesign a primer. Once I missed a nucleotide at the 2nd last position of my forward primer and it still worked.
5 bases at the end of a primer sounds like a lot, I wouldn't think it would work. Depends how long it is as well. Do you know whether it worked well for your collegue?
Thank you twister and kant0008 for the reply.
The reaction didn't work anytime before.. but it does give a smear so i was trying my best before I rethink to design the primer again...
Thanx one again..
Oh, My, I didn't notice there are FIVE mismatches. In that case, you should redesign it.
yes you should definitely redesign.
Generally, the 3'end where the polymerase starts the elongation is the most important site, regarding specificity. Actually, allele-specific PCR which is used for SNP detection utilises 2 different primers which only differ in the 3'end as suifficient discrimination. So this is very important!
Søren M. Echwald, MSc., Ph.D.
One Real-time PCR kit, which covers 38.565 genes