What factors should I consider when designing amplification primers for multiplex PCR?

There are several factors to consider when designing amplification primers for multiplex PCR. One factor to consider is that PCR primers typically range from 18 to 30 base pairs (bp) in length. Shorter primers may bind to multiple regions in the genome, potentially leading to non-target amplicons. Another factor to consider is to avoid repeats of 4 or more consecutive bases or dinucleotides as this can lead to non-specific binding. Additionally, depending on the specific application and target sequence, the length of the PCR amplicon may vary between 120-500 bp. Another factor is the melting temperature (Tm) of primers should ideally fall between 58°C and 60°C, with all primers in a reaction having a Tm within 0.5 to 1°C of one another. Tm is influenced by the base content of the primer, with higher Tm values associated with greater proportions of G and C bases compared to A and T bases. Another factor is the 3’ end of the primer should not consist of more than 3 C or G bases in the last 5 bases to ensure primer stability and binding. Lastly, The GC content of primers is recommended to be between 40% and 60%, with higher GC content preferred for templates with elevated GC content.