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: Multiplex primer design for bisulfite sequencing and 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.