Gene Synthesis by Serial Cloning of Oligonucleotides

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Abstract
A rapid and simple method of gene synthesis is presented. A gene is constructed by serial additions of individual gene fragments in the 5'' .fwdarw. 3'' direction. The vector used as the synthesis vehicle contains a unique Bsm I site at the amino terminus of the lacZ gene. Plasmid linearized with Bsm I is recircularized in vivo by oligonucleotide-directed double-strand break repair. The synthetic oligonucleotide used to "bridge" the double-strand break carries a 70- to 100-nucleotide insert which constitutes a portion of the gene along with a Bsm I site at the 3'' end that regenerates the site and allows for another consecutive round of mutagenesis to extend the gene sequence. The process is repeated until the entire gene is assembled. The method uses the .beta.-galactosidase color assay as a means of screening for correct insert lengths. The only in vitro enzymatic step necessary is a single Bsm I restriction digest of plasmid DNA. No ligation reactions are required. Only one strand of a gene sequence needs to be synthesized chemically. the gene synthesis method presented here was used to construct the human anaphylatoxin complement factor C3a gene.