期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 101, 期 44, 页码 15573-15578出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0406911101
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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 approximate to5-kb segments of DNA, followed toy 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 approximate to5-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 approximate to5-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 polyketicle product in Escherichia coli.
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