NDLI: Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster

Content Provider	PubMed Central
Author	Kodumal, Sarah J. Patel, Kedar G. Reid, Ralph Menzella, Hugo G. Welch, Mark Santi, Daniel V.
Copyright Year	2004
Abstract	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.
Related Links	http://dx.doi.org/10.1073/pnas.0406911101
Starting Page	15573
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	44
Volume Number	101
Language	English
Publisher	National Academy of Sciences
Publisher Date	2004-11-02
Access Restriction	Open
Rights Holder	National Academy of Sciences
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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