POSoligo: in vitro gene synthesis oligonucleotide software

Abstract

Abstract Oligonucleotide synthesis is crucial for molecular experiments. Bioinformatics has been applied to develop various algorithm-based tools for the in vitro synthesis of nucleotides. The primary method of synthesizing long-chain DNA molecules involves connecting short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules exhibit low mutagenesis rates, while LCR requires complementary interfaces on both ends of the two nucleic acid molecules or might alter the conformation of nucleotide chain conformation, terminating amplification. Therefore, molecular melting temperature, length, and specificity should be considered during the experimental design. Patch oligonucleotide synthesis (POS)oligo is a specialized offline tool for nucleotide fragment synthesis.. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, generating multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5′- and 3′-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.