Chemical synthesis and cloning of the human growth hormone releasing factor gene

Human growth hormone releasing factor (hGRF) gene has been synthesized and cloned. The sequence of the synthetic hGRF gene, consisting of preferred codons for expression in E. coli, was designed with the aid of computer programs. Six segments with lengths ranging from 39 to 51 nucleotides were synthesized by solid-phase phosphoramidite method. The entire gene of 141 base pairs was constructed by enzymatic ligation of all synthetic segments and then cloned into plasmid pUC-19. The positive colonies were confirmed by the screening of ampicillin resistance, inactive beta-galactosidase, and analyzing by use of restriction enzymes and dot-blot hybridization. The cloned gene was sequenced by M13 dideoxynucleotide chain termination method and proven correct.