Researchers at the J. Craig Venter Institute (JCVI), a not-for-profit genomic research organization, have published a paper describing a significant advance in genome assembly in which the team can now assemble the whole bacterial genome, Mycoplasma genitalium, in one step from 25 fragments of DNA.
Lead author Daniel G. Gibson, Ph.D. and his team published their results in the online early edition of the journal Proceedings of the National Academy of Sciences (PNAS). The work was funded by the company Synthetic Genomics Inc. (SGI).
The publication represents major improvements in the methods that the team developed and described in their January 2008 publication of the first synthesis of a bacterial genome, M. genitalium. That publication outlined how the team synthesized in the laboratory the 582,970 base pair M. genitalium genome using the chemical building blocks of DNA-adenine (A), guanine (G), cytosine (C) and thymine (T).
While this was a big advance, it took several years to come to fruition and in the end was a tedious, multi- stage process in which the team had to build the genome a quarter at a time using the bacterium Escherichia coli to clone and produce the DNA segments.
During this building process the team found that E. coli had difficulty reproducing the large DNA segments, so they turned to the yeast Saccharomyces cerevisiae. They were then able to finish creating the synthetic bacterial genome using a method called homologous recombination.
Realizing how robustly yeast performed, the team wondered if it could be used to build the entire M. genitalium genome from multiple, smaller, overlapping segments of DNA. For this study the team used DNA fragments that ranged in size from about 17,000 base pairs to 35,000 base pairs. These relatively short segments were inserted into yeast cells in one step and through the mechanism of homologous recombination were assembled into the synthetic M. genitalium genome.
Several experiments were then done to confirm that all 25 pieces of the synthetic DNA had been correctly assembled in the yeast cells, and to show that the experiment could be successfully reproduced.
The JCVI team continues to explore the capacity for DNA assembly in yeast, and the various applications of this particular method. They conjecture that a variety of combinations of DNA molecules and genetic pathways could be manufactured in yeast, in essence turning yeast into a genetic factory for specifically designed and optimized processes.