De novo gene synthesis design using TmPrime software.

Area of Science:

• Molecular Biology
• Bioinformatics
• Synthetic Biology

Background:

• De novo gene synthesis is crucial for synthetic biology and genetic engineering.
• Designing optimal oligonucleotides for gene synthesis can be complex and time-consuming.
• Existing tools may not adequately address the challenges of designing primers for both LCR and PCR-based synthesis.

Purpose of the Study:

• To present TmPrime, a novel computer program for designing oligonucleotides for de novo gene synthesis.
• To enable efficient and accurate gene synthesis using ligase chain reaction (LCR) and polymerase chain reaction (PCR) methods.
• To provide tools for sequence pooling and codon optimization to facilitate large-scale gene construction.

Main Methods:

• TmPrime divides long DNA sequences based on user-defined melting temperatures and assembly conditions.
• The software dynamically optimizes oligonucleotide length for homologous melting temperatures.
• It incorporates sequence pooling for multipool assembly and codon optimization based on organism-specific frequencies.

Main Results:

• TmPrime successfully designs oligonucleotides for LCR- and PCR-based gene synthesis.
• The program outputs melting temperatures, oligonucleotide sequences, and potential secondary structures.
• Successfully used for designing and synthesizing genes exceeding 20 kbp in total length.

Conclusions:

• TmPrime is a valuable and freely available tool for researchers involved in de novo gene synthesis.
• The software simplifies the design process, improving efficiency and accuracy in gene construction.
• TmPrime facilitates the synthesis of large and complex genes through optimized oligonucleotide design and additional functional modules.