Long oligos: direct chemical synthesis of genes with up to 1728 nucleotides

The longest oligos that can be chemically synthesized are considered to be 200-mers. Here, we report direct synthesis of an 800-mer green fluorescent protein gene and a 1728-mer Φ29 DNA polymerase gene on an automated synthesizer. Key innovations that enabled this breakthrough include conducting the synthesis on a smooth surface rather than within the pores of traditional supports, and the use of the powerful catching-by-polymerization (CBP) method for isolating the full-length oligos from a complex mixture. Conducting synthesis on a smooth surface not only eliminated the steric hindrance that would otherwise prevent long oligo assembly, but also, surprisingly, drastically reduced synthesis errors. Compared with the benchmark PCR assembly gene synthesis method, the direct long oligo synthesis method has the advantages of higher probability to succeed, fewer sequence restrictions, and being able to synthesize long oligos containing difficult elements such as unusually stable higher-order structures, long repeats, and site-specific modifications. The method is expected to open doors for various projects in areas such as synthetic biology, gene editing, and protein engineering.