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Synthetic DNA in The Lab

January 24th, 2008 Medgadget Editors News

At the J. Craig Venter Institute, scientists reproduced artificially the DNA of bacterium Mycoplasma genitalium JCVI-1.0, its genome consisting of 582,970 base pairs. This is the first time that a full sequence for life has come out of a lab. Well, surely only God knows what this technology could lead to.

The building blocks of DNA—adenine (A), guanine (G), cytosine (C) and thiamine (T) are not easy chemicals to artificially synthesize into chromosomes. As the strands of DNA get longer they get increasingly brittle, making them more difficult to work with. Prior to today’s publication the largest synthesized DNA contained only 32,000 base pairs. Thus, building a synthetic version of the genome of the bacteria M. genitalium genome that has more than 580,000 base pairs presented a formidable challenge. However, the JCVI team has expertise in many technical areas and a keen biological understanding of several species of mycoplasmas.
“When we started this work several years ago, we knew it was going to be difficult because we were treading into unknown territory,” said Hamilton Smith, M.D., senior author on the publication. “Through dedicated teamwork we have shown that building large genomes is now feasible and scalable so that important applications such as biofuels can be developed.”
The process to synthesize and assemble the synthetic version of the M. genitalium chromosome began first by resequencing the native M. genitalium genome to ensure that the team was starting with an error free sequence. After obtaining this correct version of the native genome, the team specially designed fragments of chemically synthesized DNA to build 101 “cassettes” of 5,000 to 7,000 base pairs of genetic code. As a measure to differentiate the synthetic genome versus the native genome, the team created “watermarks” in the synthetic genome. These are short inserted or substituted sequences that encode information not typically found in nature. Other changes the team made to the synthetic genome included disrupting a gene to block infectivity. To obtain the cassettes the JCVI team worked primarily with the DNA synthesis company Blue Heron Technology, as well as DNA 2.0 and GENEART.
From here, the team devised a five stage assembly process where the cassettes were joined together in subassemblies to make larger and larger pieces that would eventually be combined to build the whole synthetic M. genitalium genome. In the first step, sets of four cassettes were joined to create 25 subassemblies, each about 24,000 base pairs (24kb). These 24kb fragments were cloned into the bacterium Escherichia coli to produce sufficient DNA for the next steps, and for DNA sequence validation.
The next step involved combining three 24kb fragments together to create 8 assembled blocks, each about 72,000 base pairs. These 1/8th fragments of the whole genome were again cloned into E. coli for DNA production and DNA sequencing. Step three involved combining two 1/8th fragments together to produce large fragments approximately 144,000 base pairs or 1/4th of the whole genome.
At this stage the team could not obtain half genome clones in E. coli, so the team experimented with yeast and found that it tolerated the large foreign DNA molecules well, and that they were able to assemble the fragments together by homologous recombination. This process was used to assemble the last cassettes, from 1/4 genome fragments to the final genome of more than 580,000 base pairs. The final chromosome was again sequenced in order to validate the complete accurate chemical structure.

Press release: Venter Institute Scientists Create First Synthetic Bacterial Genome
(hat tip: Drudge Report)

Medgadget Editors

Medical technologies transform the world! Join us and see the progress in real time. At Medgadget, we report the latest technology news, interview leaders in the field, and file dispatches from medical events around the world since 2004.

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