Abstract:
The present invention provides methods of PCR-directed gene synthesis that may be used for all genes, including those with a high G+C content and/or a long sequence. The invention relates to methods of gene synthesis using overlapping oligonucleotides and polymerase chain reaction (PCR), wherein several PCR parameters, e.g., the concentration of overlapping oligonucleotides, the type of DNA polymerase used, and the number of PCR amplification cycles, are optimized. Additionally, the invention relates to oligonucleotide design that allows for increased protein expression of synthesized genes.