In this example, four amplicons are in a linear arrangement along a portion of human DNA. Each amplicon consists of an outer pair (solid arrows) and an inner nested pair (dashed arrows) of primers.
The first PCR step is a multiplex reaction containing all four pairs of outer primers. In this manner, the same DNA sample is used for all four reactions. When sample quantity is limited, this effectively increases the sensitivity of detection of each amplicon. The disadvantages are some amplicons are favored over others, there are more opportunities for non-specific amplification, and the total number of amplicons detectable by qPCR is limited by ability to distinguish the respective dyes.
The second step is four individual PCR reactions, each containing a different pair of nested primers, which gives rise to a single PCR product. Performing this second step overcomes the disadvantages of the first step multiplex PCR. Additional specificity is gained because the nested pair of primers are different sequences, giving additional priming selectivity.
Example of 2-Step PCR: Multiplex Nested PCR