A Software for Designing Oligonucleotides for PCR-Based Long DNA Synthesis

Herein we present a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). The program divides the long input DNA sequence based on the input number of oligonucleotides, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences, and potential formation of secondary structures. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp).