Parameters for Successful PCR Primer Design

  • Stephen A. BustinEmail author
  • Reinhold Mueller
  • Tania Nolan
Part of the Methods in Molecular Biology book series (MIMB, volume 2065)


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.

Key words

MIQE Oligonucleotide primers qPCR Assay design 


  1. 1.
    Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. Scholar
  2. 2.
    Taylor S, Wakem M, Dijkman G et al (2010) A practical approach to RT-qPCR-publishing data that conform to the MIQE guidelines. Methods 50:S1–S5. Scholar
  3. 3.
    Huggett JF, Foy CA, Benes V et al (2013) The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem 59:892–902. Scholar
  4. 4.
    Derveaux S, Vandesompele J, Hellemans J (2010) How to do successful gene expression analysis using real-time PCR. Methods 50:227–230. Scholar
  5. 5.
    Bustin S, Huggett J (2017) qPCR primer design revisited. Biomol Detect Quantif 14:19–28. Scholar
  6. 6.
    Latorra D, Arar K, Hurley JM (2003) Design considerations and effects of LNA in PCR primers. Mol Cell Probes 17:253–259CrossRefGoogle Scholar
  7. 7.
    Fratczak A, Kierzek R, Kierzek E (2009) LNA-modified primers drastically improve hybridization to target RNA and reverse transcription. Biochemistry 48:514–516. Scholar
  8. 8.
    Malgoyre A, Banzet S, Mouret C et al (2007) Quantification of low-expressed mRNA using 5′ LNA-containing real-time PCR primers. Biochem Biophys Res Commun 354:246–252. Scholar
  9. 9.
    Mamedov TG, Pienaar E, Whitney SE et al (2008) A fundamental study of the PCR amplification of GC-rich DNA templates. Comput Biol Chem 32:452–457. Scholar
  10. 10.
    Singh VK, Govindarajan R, Naik S et al (2000) The effect of hairpin structure on PCR amplification efficiency. Mol Biol Today 1:67–69Google Scholar
  11. 11.
    Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582. Scholar
  12. 12.
    Ruiz-Villalba A, van Pelt-Verkuil E, Gunst QD et al (2017) Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR). Biomol Detect Quantif 14:7–18. Scholar
  13. 13.
    Linhart C, Shamir R (2005) The degenerate primer design problem: theory and applications. J Comput Biol 12:431–456. Scholar
  14. 14.
    Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. BioTechniques 24:954–962PubMedGoogle Scholar
  15. 15.
    van den Berge M, Sijen T (2017) Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids. Electrophoresis 38:3155–3160. Scholar
  16. 16.
    Dieffenbach CW, Lowe TM, Dveksler GS (1993) General concepts for PCR primer design. PCR Methods Appl 3:S30–S37CrossRefGoogle Scholar
  17. 17.
    Bustin SA, Beaulieu JF, Huggett J et al (2010) MIQE précis: practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol 11:74. Scholar
  18. 18.
    Bustin SA, Benes V, Garson JA et al (2011) Primer sequence disclosure: a clarification of the MIQE guidelines. Clin Chem 57:919–921. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Stephen A. Bustin
    • 1
    Email author
  • Reinhold Mueller
    • 2
  • Tania Nolan
    • 3
  1. 1.Faculty of Health, Education, Medicine, and Social CareAnglia Ruskin UniversityChelmsfordUK
  2. 2.RM ConsultingSan DiegoUSA
  3. 3.Faculty of Medical and Human Sciences, Institute of Population HealthUniversity of ManchesterManchesterUK

Personalised recommendations