What are the limitations of multiplex PCR?

There are also various drawbacks of multiplex PCR. Firstly, there can be cases of self-inhibition among different sets of primers used in the reaction. This means that certain primers may interfere with the amplification of other target sequences, leading to inconsistent results. Additionally, multiplex PCR may not exhibit identical efficiency across different templates or target sequences. Variations in template complexity, GC content, or primer binding sites can affect the efficiency of amplification for individual targets. Multiplex PCR may display lower amplification efficiency compared to singleplex PCR reactions. This is because the presence of primer dimer sets in the same reaction can result in competition for reaction components such as dNTPs and enzymes. Multiplex PCR also requires a single set of amplification conditions for each PCR product, limiting flexibility in optimizing conditions for individual sequences.