by Christoph Weigel
In May 1984, readers of an article by Schwartz and Cantor in the prestigious journal Cell were to stumble upon pictures of DNA agarose gels that were among the lousiest of such ever published after the method was introduced in the early 70s. Why did the editors of Cell risk their reputation? Or did they, in fact?
No, they didn’t, because the readers were allowed the first glimpse at the DNA of full-length yeast chromosomes, nicely separated by length. With two simple but ingenious twists, Schwartz and Cantor had increased the power of agarose gel electrophoresis to resolve large DNA molecules by roughly 2 orders of magnitude.
Twist 1: Although the underlying physics is still debated, it is generally accepted that the movement of a linear DNA molecule (a large polyanion with distributed charges) in a homogeneous electric field is dependent on the log of its net charge (thus its length) once the molecule has adopted the optimal conformation for migration through the 3D sieve of the agarose matrix. Thus, linear DNA fragments form bands that become visible when the gel is stained after electrophoresis. Because gels with agarose concentrations below ~0.8% easily collapse in your hands and cannot be handled securely, the upper limit of resolution for linear DNA molecules in uniform electrophoresis is in the range of 30 – 50 kb. To overcome this limitation for the resolution of chromosome-sized linear DNA molecules, Schwartz and Cantor assumed that periodically changing the direction of the electrical field during electrophoresis would force the DNA molecules to reorient during each field switch, a process that would take longer for larger than for shorter molecules. Thus, the separation of large DNA molecules would then primarily depend on their reorientation time. This turned out to be the case.
Twist 2: Very large DNA molecules can migrate essentially undamaged through agarose gel matrices during electrophoresis for several hours and for distances of several centimeters, more than 100x their own length! But purifying DNA by conventional methods unavoidably results in chromosome fragmentation due to shearing forces: even the most advanced techniques available in the early 80’s failed to produce DNA molecules longer than ~40 kb. Schwartz and Cantor therefore proposed that intact chromosome-sized DNA molecules might best be purified right within the gel matrix prior to their electrophoretic separation. They embedded whole cells in agarose, trimmed the solidified agarose to obtain plugs that would fit into wells of an agarose electrophoresis gel and performed all subsequent purification steps—cell wall degradation, cell lysis, RNA and protein removal—by soaking the plugs in appropriate solutions. This, it turned out, worked fine, although DNA purification took considerably longer than by conventional means.
Technically, pulsed-field gel electrophoresis, as the technique became known, was rapidly improved by the introduction of several alternative field geometries. Pictures of chromosome-separated gels obtained were of the same quality as 'conventional' gels of small DNA fragments. It became feasible to analyze the karyotypes of species with many small chromosomes (up to ~6 Mb in size).
Among the benefits of this method was the possibility to map genes to their chromosomes by Southern hybridization, thus producing physical genome maps along with the available genetic maps. And in addition, chromosome DNA from pulsed-field gels could be purified for the making of chromosome-specific gene libraries—a prerequisite for genome sequencing in the pre-Illumina days.
In medical microbiology, the pulsed-field gel electrophoresis technique was successfully used in combination with 'rare cutter' restriction enzymes to correlate 'karyotypes' with serotypes for many of the diverse human pathogens (see here for an example). It was not a big surprise when genomic sequences later confirmed their highly mosaic and scrambled genome structures.
In the genomic era now, pulsed-field gel electrophoresis is no longer the 'gold standard' for epidemiology but it's still the method of choice for karyotyping fungal genomes. The curious STC reader can find more here.
Christoph is a lecturer in Life Science Engineering at the Hochschule für Technik und Wirtschaft, Berlin, Germany.
Schwartz DC, & Cantor CR (1984). Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell, 37 (1), 67-75 PMID: 6373014
Liu SL, & Sanderson KE (1995). Rearrangements in the genome of the bacterium Salmonella typhi. Proceedings of the National Academy of Sciences of the United States of America, 92 (4), 1018-22 PMID: 7862625