Assembling Large DNA Molecules in a Test Tube

Daniel Gibson and colleagues, previously assembled the entire 583 kilo-base-pair genome of the bacterium Mycoplasma genitalium from synthetic DNA, but the final steps of this assembly could only be accomplished in a yeast cell. However, the use of yeast cells makes the assembly process more difficult, because the foreign DNA may be toxic to the yeast.

Now the researchers modified the assembly process by directly incubating the DNA segments with three enzymes that are essential for assembly: an enzyme that chews back the DNA strands to create overlapping ends, an enzyme that synthesizes DNA and fills the gaped ends, and an enzyme that binds together the ends of the DNA fragments thus closing the gaps. The choice of the right enzymes allows the reaction to proceed entirely in a test tube in a single step, making it a faster and fully controllable process.

Author contact:

Daniel Gibson (J. Craig Venter Institute Synthetic Biology, Rockville, MD, USA)
E-mail: dgibson@jcvi.org

Abstract available online.

(C) Nature Methods press release.

Message posted by: Trevor M. D'Souza