ColabPCR: A validated Google Colaboratory Notebook for Reproducible and Precise Primer Design

The Polymerase Chain Reaction (PCR) method often has a lower success rate when amplifying specific genomic regions in eukaryotic genomes. This is frequently due to the non-specific annealing of primers at various genomic locations. To address this issue, we created ColabPCR, a program specifically designed to optimize the selection and evaluation of primers for a defined genomic region. ColabPCR refines primer length and melting temperature parameters through the utilization of Primer3 software, and it quantifies the number of potential off-target binding regions within the target genome via BLASTn analysis. Furthermore, the program facilitates the integration of restriction enzyme recognition sites at the 5’ termini of primers and provides a mechanism to confirm the absence of such sites within the intended amplification region. ColabPCR centralizes all these functionalities within a single interface, utilizing Google Colab’s computational resources to ensure high performance and accessibility without requiring local software installation. We rigorously validated ColabPCR by designing primers for promoter and terminator regions within the Daucus carota (DCARv2, DH1v3) reference genome. Our findings unequivocally demonstrate significantly enhanced success rates, particularly when primers exhibiting off-target binding are excluded from the primer design process. In summary, ColabPCR offers a user-friendly and powerful solution that simplifies and enhances the primer design and evaluation workflow, leading to increased accuracy and success in molecular biology experiments.