Multiplex-PCR Primer Set Selection


Numerous high-throughput genomics assays require the amplification of a large number of genomic loci of interest. Amplification is cost-effectively achieved using several short single-stranded DNA sequences called primers and polymerase enzyme in a reaction called multiplex polymerase chain reaction (MP-PCR). Amplification of each locus requires that two of the primers bind to the forward and reverse DNA strands flanking the locus. Since the efficiency of PCR amplification falls off exponentially as the length of the amplification product increases, an important practical requirement is that the distance between the binding sites of the two primers should not exceed a certain threshold.

Although there are numerous available PCR primer selection tools, they typically focus on single primer pair optimization and do not explicitly enforce bounds on PCR amplification length. The two tools we provide, G-POT and PRIMER-ILP, minimize the number of primers needed to amplify a set of loci while ensuring that each locus is amplified by a pair of primers located within L basepairs of each other, where L is an input parameter. G-POT is based on the “potential function” greedy algorithm described in the reference below, while PRIMER-ILP is based on integer programming. PRIMER-ILP produces optimal sets of primers, but has practical runtime only for small problem instances. G-POT is a very scalable provably-good algorithm that also produces sets of primers very close to optimal in practice.

G-POT source code

PRIMER-ILP source code

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Acknowledgment and Disclaimer

This material is based upon work supported in part by the National Science Foundation under Grants No. IIS-0546457 and DBI-0543365 and a Large Faculty Research Grant from the University of Connecticut Research Foundation. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the University of Connecticut Research Foundation.