Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome

被引：799

作者：

Gibson, Daniel G. ^{[1
]}

Benders, Gwynedd A. ^{[1
]}

Andrews-Pfannkoch, Cynthia ^{[1
]}

Denisova, Evgeniya A. ^{[1
]}

Baden-Tillson, Holly ^{[1
]}

Zaveri, Jayshree ^{[1
]}

Stockwell, Timothy B. ^{[1
]}

Brownley, Anushka ^{[1
]}

Thomas, David W. ^{[1
]}

Algire, Mikkel A. ^{[1
]}

Merryman, Chuck ^{[1
]}

Young, Lei ^{[1
]}

Noskov, Vladimir N. ^{[1
]}

Glass, John I. ^{[1
]}

Venter, J. Craig ^{[1
]}

Hutchison, Clyde A., III ^{[1
]}

Smith, Hamilton O. ^{[1
]}

机构：

[1] J Craig Venter Inst, Rockville, MD 20850 USA

来源：

SCIENCE | 2008年 / 319卷 / 5867期

关键词：

D O I：

10.1126/science.1151721

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

We have synthesized a 582,970 - base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI- 1.0, contains all the genes of wild- type M. genitalium 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 Escherichia coli. 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 Saccharomyces cerevisiae, 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.

引用

页码：1215 / 1220

页数：6

共 31 条

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