How do you design a successful multiplex PCR?

How do you design a successful multiplex PCR?


This question has been a common theme among DNAS customers since our inception 15 years ago.  Whether the multiplex is used as a molecular diagnostic or for gene enrichment libraries for NGS, the problem remains the same: multiplex is hard.  Primers that behave well in a singleplex reactions don’t seem to “play well with others”.  Fundamentally, this failure is due to two factors:  1. To prevent one PCR reaction from “taking over the PCR” (by consuming reagents and binding all the polymerase) it is essential to design all the primers to work with the same binding efficiency.  2.  A multiplex PCR is a “system” in which the parts can interact (e.g. to form undesired false amplicon artifacts) and thus the design needs to take into account the interactions among all the primers and the background genomic DNA.

Many users think that matching the Tm (2-state melting temperature) is the best method to make all the primers hybridize with the same efficiency.  However, the 2-state Tm is NOT the best metric because it neglects the competing target secondary structure.  Instead, the percent of oligonucleotide bound to target is a MUCH better metric( click here for Myth #3).  The percent bound is computed using our “multi-state coupled equilibrium model”.

Many users think that amplification artifacts such as “primer dimers” are due to the primers cross-hybridizing to each other using their 3′-ends ( click here for Myth #4).  However, primer-primer cross-hybridization is very rarely the cause of amplification artifacts.  Instead, the real mechanism by which false amplicons occur is through the hybridization of the primers to background genomic DNA (either contamination or off-target site of the genome of interest).  The chances of such primer mishybridization exponentially increases with the size of the multiplex.  To detect such false amplicons requires a tool that can detect ALL the hybridization sites for ALLthe primers and then predict all the false amplicons.  Our ThermoBLAST algorithm is the proper tool to detect these false amplicons.

For more information, download the “7 Myths of PCR Design ” (

DNA Software has helped customers at both large and small companies, to design new assays, to diagnose the issues with existing assays,  and to solve the most challenging multiplex PCR problems ranging in scale from a two-plex all the way to a 500-plex and beyond.   We’ll be addressing more of the best practices in “The Difference is in the Design” webinar series, please complete the form below to register for our next webinar.