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Multiplex PCR Primer Design


Primer and probe design is a crucial step for a successful experiment.

The rules for designing primers and probes in a digital PCR assay are similar as for a qPCR assay:


Their length should be between 18 and 25 base pairs.

The criterion that you have to carefully monitor are:

  • Percentage of GC: 40-60%
  • Primer melting temperature (Tm): ideally between 55-65°C and Tm between both primers should not differ by more than 5°C
  • G or C bases at the 3′ of the primer but not more than 2 in the last 5 bases
  • Low probability of dimer or hairpin formation


The length should not exceed 30 base pairs. Ideally 15 base pairs for optimal specificity.

The criterion that you have to carefully monitor are:

  • Percentage of GC: 20% to 80%
  • Probe melting temperature: ideally from 4 to 10°C above the primer melting temperature
  • Absence of more than 4 G repeats
  • Low probability of dimer or hairpin formation
  • Absence of G at the beginning of the probe

Sometimes, you will need to increase the Tm of the probe while keeping its size short. You can use modified bases such as locked nucleic acid (LNA) bases or peptide nucleic acid (PNA) bases. A probe with a minor groove binder (MGB) group is also an option to increase the Tm.


For further information about probe design, please refer to the following publications:

  • Design of primers and probes for quantitative real-time PCR methods. Rodríguez A, Rodríguez M, Córdoba JJ, Andrade MJ, Methods Mol Biol. 2015;1275:31-56. doi: 10.1007/978-1-4939-2365-6_3. [PMID: 25697650]

More information about MGB group, LNA, and PNA bases:

  • Locked nucleic acids in PCR primers increase sensitivity and performance. Ballantyne KN, van Oorschot RA, Mitchell RJ. Genomics. 2008 Mar;91(3):301-5. doi: 10.1016/j.ygeno.2007.10.016.[PMID: 18164179]
  • An introduction to peptide nucleic acid. Nielsen PE, Egholm M. Curr Issues Mol Biol. 1999;1(1-2):89-104. Review. [PMID: 11475704]

Tools for primer and probe designs:

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