Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene.
We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D012085:Chymosin / Q000235:genetics*; D004792:Enzyme Precursors / Q000235:genetics*; D005813:Genes, Synthetic; D008026:Ligation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010766:Phosphorylation
D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; C032443:prorennin; D012085:Chymosin