Gene manipulation of fluorescent protein through site-selective hydrolysis by Ce(IV)/EDTA.

Previously we reported that gap-site, formed in substrate DNA by using two oligonucleotide additives, was selectively hydrolyzed by Ce(IV)/EDTA. Herein this site-selective scission was used for gene manipulation, and green fluorescent protein (GFP) was converted to blue fluorescent protein (BFP). The sense strand of GFP was cleaved at predetermined site by using this system, and its upstream fragment was connected with the downstream of BFP gene by T4 DNA ligase. In this manipulation, three amino acid residues in GFP (C at the position 65, Y at 66, and T at 167) were converted to S, H, and I, respectively. The sequencing experiment confirmed that desired recombinant DNA was prepared, and the recombinant DNA was successfully expressed to emit blue fluorescence.