POSoligo software for in vitro gene synthesis

Area of Science:

• Molecular Biology
• Bioinformatics
• Synthetic Biology

Background:

• Oligonucleotide synthesis is crucial for molecular biology experiments.
• Current methods like ligase chain reaction (LCR) and polymerase chain reaction (PCR) for synthesizing long DNA molecules have limitations, including potential amplification termination and reliance on melting temperature (Tm) values.
• Designing synthetic oligonucleotides requires careful consideration of length, specificity, and potential secondary structures.

Purpose of the Study:

• To introduce POSoligo, a specialized offline bioinformatics tool for optimizing oligonucleotide synthesis.
• To enable the efficient design of contiguous long strands (COS) and short patch strands (POS) for constructing long DNA molecules.
• To demonstrate the application of POSoligo in synthesizing the SARS-CoV-2 RBD sequence.

Main Methods:

• POSoligo optimizes oligonucleotide length and specificity from a single-stranded DNA input.
• It generates COS and POS fragments with complementary ends, ensuring free 5' and 3' ends to prevent secondary structures.
• The tool facilitates ligation via LCR and PCR amplification using designed primers.

Main Results:

• POSoligo successfully designed oligonucleotides for synthesizing the linear RBD sequence of SARS-CoV-2.
• The process involved a single DNA strand, multiple POS fragments for LCR, and two primer pairs for PCR.
• This approach demonstrated a time- and cost-effective method for synthesizing long DNA molecules.

Conclusions:

• POSoligo is an effective bioinformatics tool for designing synthetic oligonucleotides for complex molecular constructs.
• It overcomes limitations of traditional methods by ensuring specific binding and preventing secondary structures without relying solely on Tm values.
• The tool offers a practical and efficient solution for producing long DNA sequences, as exemplified by the SARS-CoV-2 RBD synthesis.