Problem in Multiplex PCR - Multiplex PCR (Aug/12/2005 )

I have problem with multiplex PCR, I am using three SCAR primers (each forward and reverse) in the same PCR reaction. In my experiment, I am testing two parents (A, B) and four progenies (1,2,3,4).

If I test the parents and progenies with each primer separetely,
Both SCAR primers I and II are giving bands for parent A and for progeny 1 and 2, and
SCAR primer III (I want to use it as a control) gives bands for both parents and four progenies (which is nice).

However when I do Multiplex PCR with the above mentioned three SCAR primers, the banding pattern is as follows.
SCAR I (375 bp) = gives the same banding pattern as it gave when used alone.
SCAR II (450 bp )= Difficult to see the banding pattern (No amplification)
SCAR III (700 bp) = no band for progenies however same banding pattern for parents as it gave alone.

IF somebody can help me to solve this problem, I want that SCAR primer III and II should give the same results in multiplex PCR as they gave when used separetely.
Thank you very much for your help
Awais

Dear KA,

have you optimised the primer concentration in your multiplexed reaction??

You may need to titrate the primers for each gene to get a response that matches the single gene PCR result. If one product is more highly expressed, there will be more amplification and thus more reagents being used to create that product thereby limiting the resources for the more lowly expressed genes.

Hope this helps,

AussieUSA.

Thanks Aussie for reply.
This is not clear to me that why the SCAR primer III is giving bands only for parents and not for the progenies in multiplex reaction, whereas it is giving bands for parents as well as progenies when used individually. I would have expected no band at all for parents and progenies if multiplex was not working.
Awais, Zurich

I did a multiplex PCR on a bacterial endotoxin. It turned out to be very temperature sensitive. It only gave all the bonds at the temperature 56.3 C!

So my advice would be to optimize the reaction, regarding the annealing temperature. And also, as AussieUSA says, the primer concentrations.