PCR: multiplex ok, single reactions not - microsatellite analysis (Jul/07/2006 )
A similar mesasge has been posted before, but I left out an important sentence, so it ended up quite meaningless.
Dear anyone interested
I am currently testing parameters for multiplex PCR for subsequent microsatellite analysis on a sequencer. I have been running my 12 loci with different MgCl2 concentrations, alone and as multiplex reactions. In this early phase, the products are run on an agarose gel.
The multiplex reactions (with three primer-pairs) keep resulting in quite strong bands on the gel, but when I run the primer-pairs alone, the bands rarely show.
This is weird! All other paramters are the same, except that the primers are diluted in TE buffer, so there is more TE buffer in the multiplex reactions, due to more primers. This, though, I thought would weaken the reaction since TE binds MgCl2.
Anyone got an idea whats going on here?
Best Regards Vegard
Worst scenario is that the band corresponding to multiplex PCR that you saw could be an artifact from hetero- or homo-dimer formation.
Multiplex PCR tend to have this problem quite frequently. That's why it's a big headache to try to design primers (all of the sets) that will not interact with one another, but to the template(s) of interest.
You'll also have to do lots of optimization.
Can't help much but hope the following will assist you in anyway.
Broude et al 2001. Multiplex allele-specific target amplification based on PCR suppression. PNAS 98(1):206-211
A Primer Design Software for MpxPCR (Free software)
Thank you very much. The reactions seem to be working better now, but these links will be helpful in the future anyway.