what problems arise when one sets up a multiplex pcr?

Answer 1

There are lots of different problems. For starters, you have to find primers that can co-exist well. They need to have similar melting temps so similar length and GC content or if not similar in length appropriate GC content to obtain similar annealing temps. Then, you have to make sure the primers do not contain sequences that would cause them to hybridize to one another. Once you think you have the kinks worked out with your primer-design, you have to worry about the length of your PCR products. If you have some products that are much longer than others, it means the optimal extension time will be different for the different products and so you will have to find an extension time that works for both.

Basically, multiplex PCR gives you a lot more optimization headaches than a single primer pair will.

Answer 2

The short list:

If one primer set works better then the other (or all the others), it will out compete the others for all the reagents and you'll get lots of one product and little to nothing from the others.

A primer from one set might find a region of homology with a primer from another set just large enough to form what we refer to as primer-dimer. This aberrant product will also quickly burn up the one primer from each set and prevent or greatly inhibit the formation of the products that would have been formed by the other 2 sets.

I'm finding it a real challenge to multiplex with 3-4 primer sets, but I've heard of people doing as many as 48sets.