Summary: Designing PCR primers for amplifying regions of eukaryotic genomes is a complicated task because the genomes contain a large number of repeat sequences and other regions unsuitable for amplification by PCR. We have developed a novel k-mer based masking method that uses a statistical model to detect and mask failure-prone regions on the DNA template prior to primer design. We implemented the software as a standalone software primer3_masker and integrated it into the primer design program Primer3. Availability and implementation: The standalone version of primer3_masker is implemented in C. The source code is freely available at https://github.com/bioinfo-ut/primer3_masker/ (standalone version for Linux and macOS) and at https://github.com/primer3-org/primer3/ (integrated version). Primer3 web application that allows masking sequences of 196 animal and plant genomes is available at http://primer3.ut.ee/. Contact: [email protected]. Supplementary information: Supplementary data are available at Bioinformatics online.

MOTIVATION: The concentration of pathogen DNA in biological samples is often very low. Therefore, the sensitivity of diagnostic tests is always a critical factor. RESULTS: We have developed a novel computational method that identifies species-specific repeats from microbial organisms and automatically designs species-specific PCR primers for these repeats. We tested the methodology on 30 randomly chosen microbial species and we demonstrate that species-specific repeats longer than 300 bp exist in all these genomes. We also used our methodology to design species-specific PCR primers for 86 repeats from five medically relevant microbial species. These PCR primers were tested experimentally. We demonstrate that using species-specific repeats as a PCR template region can increase the sensitivity of PCR in diagnostic tests. AVAILABILITY AND IMPLEMENTATION: A web version of the method called MultiMPrimer3 was implemented and is freely available at (http://bioinfo.ut.ee/multimprimer3/).

UNLABELLED: The determination of annealing temperature is a critical step in PCR design. This parameter is typically derived from the melting temperature of the PCR primers, so for successful PCR work it is important to determine the melting temperature of primer accurately. We introduced several enhancements in the widely used primer design program Primer3. The improvements include a formula for calculating melting temperature and a salt correction formula. Also, the new version can take into account the effects of divalent cations, which are included in most PCR buffers. Another modification enables using lowercase masked template sequences for primer design. AVAILABILITY: Features described in this article have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer) of Primer3. Also, a modified version is compiled under the name of mPrimer3 which is distributed independently. The web-based version of mPrimer3 is available at http://bioinfo.ebc.ee/mprimer3/ and the binary code is freely downloadable from the URL http://bioinfo.ebc.ee/download/.