Parameters for Successful PCR Primer Design

Methods Mol Biol. 2020:2065:5-22. doi: 10.1007/978-1-4939-9833-3_2.


Primers are critical components of any PCR assay, as they are the main determinants of its specificity, sensitivity, and robustness. Despite the publication of numerous guidelines, the actual design of many published assays is often unsound: primers lack the claimed specificity, they may have to compete with secondary structures at their binding sites, primer dimer formation may affect the assay's sensitivity or they may bind only within a narrow temperature range. This chapter provides simple guidance to avoid these most common issues.

Keywords: Assay design; MIQE; Oligonucleotide primers; qPCR.

MeSH terms

  • Binding Sites / genetics
  • DNA Primers / chemistry*
  • DNA Primers / genetics
  • Limit of Detection
  • Nucleic Acid Conformation
  • Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Temperature


  • DNA Primers