In-Silico Automated Allele-Specific Primer Design for Loop-Mediated Isothermal Amplification

Primers carry unique genetic information that allows them to be used as specific probes in various applications in the field of diagnostics and particularly in cancer, where the need for accurate treatment selection is crucial. Rapid and affordable detection of cancer specific targets, such as allele-specific single-nucleotide mutational changes, are of great need to improve treatment efficacy and guide clinical use. Detecting these mutations in isothermal conditions creates the opportunity to develop cost-efficient diagnostic platforms for cancer treatment. In this paper, a novel python script was constructed to develop a simple and personalised software tool that could automate the design of allele-specific primers in isothermal conditions using design parameters, such as free energies and annealing temperatures. The scripts' primers were compared to manually designed probes that were experimentally tested on a variant (ESR1 p.E380Q), commonly present in metastatic breast cancer, showcasing the applicability of the method and the potential for the script to be used as part of an automated software to design allele-specific assays for Lab-on-Chip platforms in cancer diagnostics.