Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster.
To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of approximately 5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440-15445]. The major current impediment to the success of this tactic is the difficulty of building the approximately 5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp "synthons" with low error frequency by automated PCR-based gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon approximately 5-kb segments by using only three endonucleases and "ligation by selection." These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.
Kodumal
Sarah J
SJ
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545, USA.
Patel
Kedar G
KG
Reid
Ralph
R
Menzella
Hugo G
HG
Welch
Mark
M
Santi
Daniel V
DV
D001483:Base Sequence; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008722:Methods; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D048630:Polyketide Synthases / Q000235:genetics*
D004269:DNA, Bacterial; D048630:Polyketide Synthases