Introduction

MFEprimer Web Server 4.0 #

Since its launch in 2009, the MFEprimer Web Server has been in continuous operation for 16 years. We remain committed to maintaining this platform as a sustainable, long-term resource for the global research community.

Beginning with version 4.0, the server has evolved into a comprehensive platform for PCR primer quality assessment and design. Recently, we introduced MP-Ref, a multiplex PCR primer design tool based on reference genomes. In addition, a new module, MP-Methy, dedicated to primer design for DNA methylation studies, is under active development and currently available for beta testing. A detailed overview of the available tools is provided below.

Quality Control Tools #

  • Specificity Check: Verifies primer specificity against multiple background databases to minimize off-target amplification.
  • Dimer Check: Evaluates the potential formation of primer–dimer structures that could compromise PCR performance.
  • Hairpin Check: Assesses the likelihood of hairpin structures that may reduce amplification efficiency.
  • Coverage Analysis for Microbes: Examines primer coverage across microbial strains to identify potential performance issues.

Primer Design Tools #

  • MP-Ref: Multiplex PCR primer design for reference genomes. This enhanced tool supports a broad range of applications and streamlines primer design for complex multiplex PCR experiments.
  • MP-Methy: Built upon MP-Ref, this tool is specifically tailored for methylation primer design. It enables primer design for bisulfite sequencing of bisulfite-treated DNA and for methylation-specific PCR, facilitating research in DNA methylation and epigenetics.

Miscellaneous Tools #

  • SubSeq: Enables retrieval of subsequences from FASTA databases for downstream analyses such as BLAST searches and GC-content evaluation.

History #

  • 2019, MFEprimer-3.0: quality control for PCR primers.
  • 2012, MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.
  • 2009, MFEprimer-1.0: multiple factor evaluation of the specificity of PCR primers.

Contact #

For bug reports or suggestions, please contact us via email.