Combination of DNA single strand synthesis with PCR to construct mung bean trypsin inhibitor gene

This paper using the synthesis of mung bean trypsin inhibitor gene as an example, presented a new method for gene synthesis. The principle of the method was based on the combination of the single strand strategy and the PCR technique. The synthesis was very simple, convenient and rapid. The mung bean trypsin inhibitor is a protein composed of 72 amino acid residues. Its amino acid sequence has been determined, but the DNA sequence of gene still unknown. The synthetic mung bean trypsin inhibitor gene was 248 bp in length. It contains the encoded sequence, the start and stop codons, the restriction sites of EcoRI and BamHI at both ends, The codon selection of the synthetic gene was carried out according to the codon usage of other plant protease inhibitor gene or plant gene. The synthetic double-stranded DNA was digested with EcoRI and BamHI or PstI first, then cloned into plasmid pUC19. The synthetic gene was proved to be correct by the restriction map and the sequence analysis using the dideoxy-mediated chain termination method.