Abstract
The design of oligonucleotides with uniform hybridisation temperatures is essential for successful gene synthesis. However, current computational tools for oligonucleotide design face significant limitations, including difficulties in processing long DNA sequences, poor adaptability to specific experimental conditions, limited control over oligonucleotide length, and challenges in minimising spurious dimer formation. To address these issues, we developed CertPrime (https://oligodesign.bifi.es), an innovative tool designed for scalable and efficient handling of long DNA sequences. CertPrime enables precise customisation of experimental parameters, provides flexibility to limit the maximum oligonucleotide length, and generates designs with reduced deviations in melting temperatures across overlapping regions compared to existing tools. We experimentally compared CertPrime designs with benchmark design methods for a complex DNA sequence and found that CertPrime designs led to more efficient gene assembly, significantly reducing the occurrence of non-specific bands. These results make CertPrime a powerful and versatile tool for oligonucleotide design in gene synthesis applications.