Primer design - GC clamp - (Aug/06/2009 )
I'm using an Internet freely available software to design primers. The tool would then tell me if the primer is nicely designed or not. In some cases I get the warning saying ''There are more than 3 G's or C's in the last 5 bases''. What would happen if I do have the primer in that case?
Because G's and C's bind more strongly than A and T, having too many of them at the end of a primer can cause the Tm to increase and make it so that the GC clamp of the primer is too strong, disallowing it from denaturing at the appropriate temperature. Having 1 or 2 G's or C's at the end of the primer is ideal, becuase it allows for strong binding of the primer to the cDNA, making it more specific, without binding too strongly.
Hope this helps!
here is a Link
I prefer to use Primer3, an internet free softeware as well. It will pick a good primer for you.
Thanks alot guy for the tips!
altobarn on Aug 6 2009, 08:49 AM said:
As far as I know, having a high GC content facilitates tight binding of the oligo to the template but also increases the possibility of mispriming.
May I know, which software are you using to check your primers? I use NetPrimer from Primer Biosoft: http://www.premierbiosoft.com/netprimer/index.html
The problem with high GC content at the 3' end is that it makes mispriming quite possible. I try to design primers with a final G or C, or perhaps two, but not three or more. Most important is to avoid strong hairpins with 5' overhangs, or strong primer-dimers with 5' overhangs.
The 3' end affects both specificity and product yield . GC poor 3' end would generally enhance specificity at the cost of product yield . That's why there should be a balance between AT and GC content at the 3' end. High GC content at the 3' end decreases specificity. GC rich 3' ends are known as " sticky ends" . Remember that annealing of only the last few nucleotides of the primer to the template DNA is more than sufficient for extention to occcur.
Hope this helps.
This question about the GC clamp on 3' ends of primers interests me a lot.
Right now, I'm using Primer 3 to pick primers from a sequence and in the GC clamp parameter field, I've chosen GC clamp equal to 0, 1 or 2 and picked primers for each situation (everything else equal). The resulting reverse primers were different for each and only the forward primer resulting from a situation of a GC clamp = 0 was different from the others.
I've had some problems trying to amplify this locus with the original primers designed for it. They're supposed to work in a conserved region (an exon), but the amplifications I got were never consistent when it comes to the amplified band intensity. So I decided to try and pick other primers more inside the exons flanking my target sequence, in hopes of having working primers that might not be subjected to any putative mutation.
So, I wonder if the primers picked with GC clamp = 0 will force a less specific amplification, but on the other had will increase the changes of yielding any product at all, specially to proceed to sequencing afterwards?
What are your opinions on this?
Listening to: Interpol - Say Hello To The Angels
I don't think there is anything wrong with primers having AT rich sequences at the 3' end, but you should be aware that high AT regions (in primers or template) may require lower extension temperatures than normal (extension, not annealing, which is a separate issue). Whatever works. Design and order a few different ones, they are now cheap enough that it is not worth your time to try and reject ones, then need to wait to try a second set.