Size matters! That is true also for molecular biology. Many of the techniques used in the laboratories have their limitations, mainly to size. Just like resolving the DNA on agarose gel. Standard electrophoresis is fine if you need to look at fragments no bigger than ~10 kb. But what if you are interested in bigger chunk of the genome or even all of it?

The solution came in 1983 by Schwartz and Cantor, who described Pulse Field Gel Electrophoresis (PFGE) and even gave the schematic drawing, so you could build your own apparatus (ah! good old days of DIY science). PFGE allows separating large fragments of DNA, i.e. yeast chromosomes. Interestingly, the technique was not understood at the beginning. The duet proposed that the DNA, forms a coil that reorients itself under the electric field, so that the long arm of the coil is parallel to the field. Thus, by changing the angle of the field direction, DNA will be reorienting itself and thus going across the gel in a zigzag mode:

The device has been subsequently optimized, so it looks more like this today:

With every new appliance, comes a subsequent flow of ideas how it may be used and the amount of derived applications increase almost exponentially. PFGE became extremely useful tool for many experiments. Cloning large fragments of DNA to YACs, chromosome restriction mapping, chromosome counting, DNA double strand break assay – PFGE is the key element to all of these. It also became a golden standard for epidemiology, allowing quick genotyping of pathogenic bacterial strains (genetic fingerprints), so that you may know if the patient has drug-resistant or highly virulent strains.

But as time goes by and new techniques are developed, the old ones face twilight and eventually fade away. Take Southern Blot. Once common, nowadays nobody really uses it, since DNA sequencing became really cheap (£3/kb). Is the same fate awaiting PFGE? Nowadays, nobody bothers with building their own machine and the only provider of the apparatus is Bio-Rad (good luck if you want your machine to be serviced – it took me 7 months before they finally admitted that they do not know what the problem is!). Therefore, would the lack of competition, as well as the appearance of novel methods, eventually send PFGE to a museum shelf? It may not be so quick…

Let’s consider DNA damage. It commonly happens in our cells. One type of DNA damage, called Double Strand Break (DSB) is potentially dangerous – any mismatch during DNA repair in the coding region can have mutagenic effect, even leading to cancer development! In 2015, the Nobel Prize in Chemistry was awarded to three scientists who studied the mechanisms of DNA repair . Ionizing radiation, water deficit, reactive oxygen species – all of these may lead to DSB (Fig. 3).

It is therefore necessary to have a good method of detecting DSB and PFGE is one of the best. Of course, there are other methods, such as Comet assay, but the advantage of PFGE is that you can actually see the fragmented DNA, isolated from the whole organisms (Comet assay works best with single cells, which may be tricky to isolate). Another methods exist such as Antibody Assay, which detect phosphorylation of one of histone proteins (H2A, to be precise) – the reaction that occurs after DSB in most organisms. Although H2A histones are conserved in most organisms, there are exceptions. One particular is bdelloid rotifer – an extremophile which can survive severe water loss and, subsequently high rate of DSB. Bdelloid H2A histones have unusual sequence and do not react with antibodies used for typical screening. It is also hard to isolate single cells, as their tissues are mostly syncytial (that is, multiple nuclei share common cytoplasm). Thereby, the only method detecting DSBs in this organism is PFGE.

Let’s have a look at the video which combines a detailed info about both DSB and PFGE:

HQ slideshow:

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Link to Behance presentation:

https://www.behance.net/gallery/32758367/PFGE-Animation