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bioRxiv · 10.1101/2025.04.29.650686

CertPrime: a new oligonucleotide design tool for gene synthesis

Luna-Cerralbo, D.·Serrano, A.·Blasco-Machin, I.·Hamdan, F.·Martinez-Olivan, J.·Broset, E.·Bruscolini, P.
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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 experimen- tal conditions, limited control over oligonucleotide length, and challenges in minimising spurious dimer formation. To address these issues, we devel- oped CertPrime, an innovative tool designed for scalable and efficient han- dling of long DNA sequences. CertPrime enables precise customisation of experimental parameters, provides flexibility to limit the maximum oligonu- cleotide 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 meth- ods for a complex DNA sequence and found that CertPrime design 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. HighlightsO_LICertPrime improves oligonucleotide design by minimising melting-temperature deviations and spurious dimer formation. C_LIO_LIThe new tool enhances gene-synthesis efficiency, allowing a precise con- trol over oligonucleotide lengths and experimental parameters. C_LIO_LICertprime is a scalable tool capable of designing over 99% of the human genome with optimized sequence assembly C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/650686v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@2032aaorg.highwire.dtl.DTLVardef@1de509borg.highwire.dtl.DTLVardef@7e2148org.highwire.dtl.DTLVardef@bb3dd2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Luna-Cerralbo, D., Serrano, A., Blasco-Machin, I., Hamdan, F., Martinez-Olivan, J., Broset, E., Bruscolini, P.. 2025-04-29. CertPrime: a new oligonucleotide design tool for gene synthesis. https://doi.org/10.1101/2025.04.29.650686

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