[Total synthesis of long DNA sequences]
>>Published online before print October 20, 2004
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0406911101
Biochemistry
Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster
Sarah J. Kodumal, Kedar G. Patel, Ralph Reid, Hugo G. Menzella, Mark Welch, and Daniel V. Santi *
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545
Communicated by Robert M. Stroud, University of California, San Francisco, CA, September 17, 2004 (received for review July 19, 2004)
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 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 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 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.<<
Possible trickle applications here?
Cheers, Tuck |
