University of Tartu
Department of Bioinformatics


Multimprimer3 is automated web-based primer design tool for primer design from bacterial repetitive DNA sequences
    Multimprimer3 designs detection PCR primers for bacteria. There is two options how to use Multimprimer3.
  1. User knows the names of bacterial species/genomes which he/she wants to detect (with one primer pair and in one sample). These names can be selected from given list (the strains, species and genera in our local database are given here). Also, user can choose non-target bacterial species/genomes that may occur in the sample. Additionally, human genomic sequence can be chosen as non-target genome (by inserting checkmark to checkbox 'Homo sapiens as non-target genome'). Currently, the genomic sequences are retrieved from NCBI (only completed genomes are retrieved). The email address may be inserted in case of time consuming tasks (if many bacterial genomes and/or human genomic sequence is chosen). Finally, general primer design options (flags of primer3) can be specified.
      Thereafter, the program looks for 3 target-specific repetitive DNA sequences that
    1. are present each at least as two different copies in each target genome (if no repetitive sequence is found then target-specific sequence with one copy is considered)
    2. not present in non of the non-target genomes
      After that queried number of
    • PCR primers are designed to consensus sequence of each repetitive sequences with primer3
    • Target-specificity of designed primers is checked with fastagrep (primers must not amplify sequences from non-target genomes)
    Primers are not assessed with any score; primer pairs are ought to be equally well working. User gets DNA sequences of PCR primers and PCR products, genomic positions of repetitive sequences which are used for primer design and some additional data about users query and primer design.

  2. User has his/her own (repetitive) sequence. The sequence must be in FASTA format. If there is more than one sequence primers will be designed to the consensus sequence composed from inserted sequences. Principally, the process for finding PCR primers for the repeat is the same as described above. The uniqueness of inserted sequence(s) is (are) checked against selected non-target genomes and the uniqueness of designed primers is examined.

some examples how to use Multimprimer3:

Two examples of usage the Multimprimer3 are presented in MANUAL