Complete Chemical Synthesis, Assembly, and Cloning of a <i>Mycoplasma genitalium</i> Genome
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- Daniel G. Gibson
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Gwynedd A. Benders
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Cynthia Andrews-Pfannkoch
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Evgeniya A. Denisova
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Holly Baden-Tillson
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Jayshree Zaveri
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Timothy B. Stockwell
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Anushka Brownley
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- David W. Thomas
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Mikkel A. Algire
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Chuck Merryman
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Lei Young
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Vladimir N. Noskov
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- John I. Glass
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- J. Craig Venter
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Clyde A. Hutchison
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
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- Hamilton O. Smith
- The J. Craig Venter Institute, Rockville, MD 20850, USA.
書誌事項
- 公開日
- 2008-02-29
- DOI
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- 10.1126/science.1151721
- 公開者
- American Association for the Advancement of Science (AAAS)
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説明
<jats:p> We have synthesized a 582,970–base pair <jats:italic>Mycoplasma genitalium</jats:italic> genome. This synthetic genome, named <jats:italic>M. genitalium</jats:italic> JCVI-1.0, contains all the genes of wild-type <jats:italic>M. genitalium</jats:italic> G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted “watermarks” at intergenic sites known to tolerate transposon insertions. Overlapping “cassettes” of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb (“1/8 genome”), and 144 kb (“1/4 genome”), which were all cloned as bacterial artificial chromosomes in <jats:italic>Escherichia coli</jats:italic> . Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast <jats:italic>Saccharomyces cerevisiae</jats:italic> , then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments. </jats:p>
収録刊行物
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- Science
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Science 319 (5867), 1215-1220, 2008-02-29
American Association for the Advancement of Science (AAAS)
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詳細情報 詳細情報について
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- CRID
- 1361137046481016448
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- ISSN
- 10959203
- 00368075
- https://id.crossref.org/issn/00368075
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- データソース種別
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- Crossref