Selecting Specific PCR Primers with MFEprimer

  • Wubin Qu
  • Chenggang ZhangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1275)


Selecting specific primers is crucial for polymerase chain reaction (PCR). Nonspecific primers will bind to unintended genes and result in nonspecific amplicons. MFEprimer is a program for checking the specificity of PCR primers against the background DNA. In this chapter, we introduce: (1) the factors that affect the specificity of primers; (2) the principle of MFEprimer and its settings; (3) how to use the MFEprimer to examine the specificity of primers.

Key words

PCR Primer specificity MFEprimer 



This work was supported by the National Basic Research Project (973 program) (2012CB518200), the General Program (31371345, 30900862, 30973107, 81070741, 81172770) of the Natural Science Foundation of China, the State Key Laboratory of Proteomics of China (SKLP-O201104, SKLP-K201004, SKLP-O201002), and the Special Key Programs for Science and Technology of China (2012ZX09102301-016).


  1. 1.
    Qu W, Shen Z, Zhao D et al (2009) MFEprimer: multiple factor evaluation of the specificity of PCR primers. Bioinformatics 25(2):276–278CrossRefPubMedGoogle Scholar
  2. 2.
    Qu W, Zhou Y, Zhang Y et al (2012) MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity. Nucleic Acids Res 40(W1):W205–W208CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Lexa M, Horak J, Brzobohaty B (2001) Virtual PCR. Bioinformatics 17(2):192–193CrossRefPubMedGoogle Scholar
  4. 4.
    Lexa M, Valle G (2003) PRIMEX: rapid identification of oligonucleotide matches in whole genomes. Bioinformatics 19(18):2486–2488CrossRefPubMedGoogle Scholar
  5. 5.
    Boutros PC, Okey AB (2004) PUNS: transcriptomic- and genomic-in silico PCR for enhanced primer design. Bioinformatics 20(15):2399–2400CrossRefPubMedGoogle Scholar
  6. 6.
    Andreson R, Kaplinski L, Remm M (2007) Fast masking of repeated primer binding sites in eukaryotic genomes. Methods Mol Biol 402:201–218CrossRefPubMedGoogle Scholar
  7. 7.
    Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    SantaLucia J Jr (2007) Physical principles and visual-OMP software for optimal PCR design. Methods Mol Biol 402:3–34CrossRefPubMedGoogle Scholar
  9. 9.
    SantaLucia J, Hicks D (2004) The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33:415–440CrossRefPubMedGoogle Scholar
  10. 10.
    Onodera K, Melcher U (2004) Selection for 3′ end triplets for polymerase chain reaction primers. Mol Cell Probes 18(6):369–372CrossRefPubMedGoogle Scholar
  11. 11.
    Miura F, Uematsu C, Sakaki Y et al (2005) A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3′-end subsequences. Bioinformatics 21(24):4363–4370CrossRefPubMedGoogle Scholar
  12. 12.
    SantaLucia J (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor hermodynamics. Proc Natl Acad Sci U S A 95(4):1460–1465CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Lindblad-Toh K, Wade CM, Mikkelsen TS et al (2005) Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438(7069):803–819CrossRefPubMedGoogle Scholar
  14. 14.
    Zhou Y, Qu W, Lu Y et al (2011) VizPrimer: a web server for visualized PCR primer design based on known gene structure. Bioinformatics 27(24):3432–3434CrossRefPubMedGoogle Scholar
  15. 15.
    Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302(1):205–217CrossRefPubMedGoogle Scholar
  16. 16.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Andreson R, Möls T, Remm M (2008) Predicting failure rate of PCR in large genomes. Nucleic Acids Res 36(11):e66CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Rychlik W (1995) Priming efficiency in PCR. Biotechniques 18(1):84–86, 88–90PubMedGoogle Scholar
  19. 19.
    Kent WJ (2002) BLAT—the BLAST-like alignment tool. Genome Res 12(4):656–664CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Panjkovich A, Melo F (2005) Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics 21(6):711–722CrossRefPubMedGoogle Scholar
  21. 21.
    von Ahsen N, Wittwer CT, Schutz E (2001) Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg2+, deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. Clin Chem 47(11):1956–1961Google Scholar
  22. 22.
    Crothers DM, Zimm BH (1964) Theory of the melting transition of synthetic polynucleotides: evaluation of the stacking free energy. J Mol Biol 9:1–9CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Beijing Institute of Radiation Medicine, State Key Laboratory of ProteomicsCognitive and Mental Health Research Center of PLABeijingChina

Personalised recommendations