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Home » , » PerlPrimer open-source PCR primer design.

PerlPrimer open-source PCR primer design.

PerlPrimer is a free, open-source GUI application written in Perl that designs primers for standard PCR, bisulphite PCR, real-time PCR (QPCR) and sequencing. It aims to automate and simplify the process of primer design.

PerlPrimer's current features include the following:

  • Calculation of possible primer-dimers
  • Retrieval of genomic or cdna sequences from Ensembl (including both sequences automatically for QPCR)
  • Ability to BLAST search primers using the NCBI server or a local server
  • Results can be saved or optionally exported in a tab-delimited format that is compatible with most spreadsheet applications.
  • ORF and CpG island detection algorithms
  • Ability to add cloning sequences to primers, automatically adjusted to be in-frame
  • QPCR primer design without manual intron-exon boundary entry

PerlPrimer calculates primer melting temperature using J. SantaLucia's extensive nearest-neighbour thermodynamic parameters.


To adjust for the salt conditions of the PCR, PerlPrimer uses the empirical formula derived by von Ahsen, et al. (2001) and allows the user to specify the concentration of Mg2+, dNTPs and primers, or use standard PCR conditions. The result is a highly accurate prediction of primer melting temperature, giving rise to a maximum yeild of product when amplified.

PerlPrimer is written in Perl and Perl/Tk. In addition, for QPCR functionality PerlPrimer uses the open-source Spidey executable from NCBI, and restriction enzyme data from the REBASE project is used when adding cloning sites. The program is designed to be cross-platform compatible and has been developed and tested on both Microsoft Windows and GNU/Linux-based operating systems. Users have also reported success using the program under Mac OS X.

Please cite the reference below if this program is useful.

Download PerlPrimer v1.1.21

PerlPrimer for Debian Linux
(May not be up-to-date for recent releases)

Perlprimer source (.bz2 archive) (~350Kb)
Perl script as above in a .tar.bz2 archive for Linux, MacOSX and other *nix users

Source installation instructions

Users of UNIX-based operating systems can extract .bz2 archives using the tar utility:

$ tar xjvf perlprimer-1.1.21.tar.bz2

Many distributions of Linux/Unix should come with Perl, Perl/Tk and libwww-perl packages. Provided Perl is installed, running PerlPrimer ($ perl perlprimer.pl [file to open]) will list which modules are not installed and are required. If these are not present in your distribution, please download and install from the links below.

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