Primer3

Primer3 is an open-source software tool for designing primers in a polymerase chain reaction (PCR), a fundamental molecular biology technique with a wide range of applications, including DNA cloning and sequencing, functional gene analysis, disease diagnosis, genotyping, and genetic variant discovery. Reliable primer design is critical for successful PCR, and Primer3 has been a trusted tool for this purpose for over a decade, often used in high-throughput genomics applications.

Over the years, Primer3 has undergone several improvements and enhancements that have expanded its functionality and made it even more reliable and versatile. In this article, the authors describe Primer3's current capabilities, emphasizing recent improvements.

One of the most significant enhancements is incorporating more accurate thermodynamic models in the primer design. This improvement results in improved melting temperature prediction and reduced likelihood of primers forming hairpins or dimers. Additionally, Primer3 now offers more precise control of primer placement, motivated partly by opportunities to use whole-genome sequences to improve primer specificity.

Ease of use has also been improved, with new features such as saving and reusing parameter settings and ensuring that individual primers are not used in more than one primer pair. The core code has been made more modular, and cleaner programming interfaces are now available to facilitate integration with other software applications.