Total Synthesis of a Gene

Key words

gene synthesis - oligonucleotides - nucleotide chemistry - DCC coupling

Significance

The entire gene encoding the precursor of the E. coli tyrosine tRNA was synthesized from protected mononucleotides. The final DNA fragment consists of 207 base-pairs and also includes a promotor region as well as a termination-inducing element. After successful cloning of this duplex, transcription was demonstrated in E. coli.

Comment

First, 26 distinct oligonucleotides were synthesized using cycles of DCC-mediated coupling and basic hydrolysis to release the free 3′-OH. Remaining protecting groups of the 9- to 13-mers were cleaved and a radioactive phosphate was installed enzymatically at the 5′-OH. Ligase-mediated couplings of four to six oligonucleotides afforded six different double-stranded duplexes, which were further ligated to yield the final gene.