Welch M

References (1)

Title : Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster - Kodumal_2004_Proc.Natl.Acad.Sci.U.S.A_101_15573
Author(s) : Kodumal SJ , Patel KG , Reid R , Menzella HG , Welch M , Santi DV
Ref : Proc Natl Acad Sci U S A , 101 :15573 , 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 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.
ESTHER : Kodumal_2004_Proc.Natl.Acad.Sci.U.S.A_101_15573
PubMedSearch : Kodumal_2004_Proc.Natl.Acad.Sci.U.S.A_101_15573
PubMedID: 15496466
Gene_locus related to this paper: sacer-ery3