What factors are critical for multiplex PCR?
What factors are critical for multiplex PCR?
All primer pairs used in multiplex PCR should have similar priming efficiencies for their target DNA. This can be achieved by using primers with nearly identical optimum annealing temperatures.
When designing primers, pay special attention the following parameters:
-Homology with the target nucleic acid sequence
-Length
-GC content
-Concentration
-Primer homology (primers should not have homology either internally or with one another, especially at the 3' ends)