Last Month's Most Accessed Feature: New gel clicks into place: To separate DNA by slab gel electrophoresis

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  • Published: Oct 4, 2017
  • Categories: Electrophoresis
thumbnail image: Last Month's Most Accessed Feature: New gel clicks into place: To separate DNA by slab gel electrophoresis

New gel clicks into place: To separate DNA by slab gel electrophoresisPolyacrylamide and agarose

Slab gel electrophoresis may be decades old but there are still only two choices of gel for scientists wanting to separate DNA: polyacrylamide and agarose. Now, a team of Italian and German scientists have come up with a third option that offers several advantages over the other two.

One problem with polyacrylamide gels is that they are often produced by exposing the acrylamide monomers to ultraviolet (UV) light. This prevents fluorescent compounds that emit light when they bind to DNA from being incorporated into the gel, because the UV light tends to deactivate them. Instead, the fluorescent compounds have to bind to the DNA prior to electrophoresis, which can have a detrimental effect on the separation process.

Agarose gels don’t suffer from this limitation, because they aren’t produced through exposure to UV light, but they aren’t very good at separating small DNA strands and so tend to be restricted to longer strands. At the moment, then, there’s no effective way to separate small DNA strands in a gel containing fluorescent compounds.

Click chemistry

This was the oversight that a team of chemists from the Institute of Chemistry of Molecular Recognition in Milan, Italy, and Agilent Technologies in Germany decided to remedy by developing a novel hydrogel. They did this by mixing together two polymers containing reactive functional groups poly(dimethylacrylamide) functionalized with an alkyne moiety and poly(ethylene glycol) bis-azide bearing azido groups at both ends and then adding copper sulphate as a polymerization catalyst. This produces a classic click chemistry reaction, in which the copper snaps together the alkyne and azido groups on each monomer, and so the chemists refer to the resultant polymer hydrogel as a click gel.

Because this polymerization process doesn’t require UV light, fluorescent compounds can be incorporated into the click gel. As a first test of their fluorescent compound-containing click gel, the chemists used it to separate a DNA ladder extending from 100 base pairs (bp) to 1500bp by electrophoresis. They compared it with a conventional polyacrylamide gel and a DNA ladder exposed to the fluorescent compound before separation.

This test was conducted using Agilent’s TapeStation, an automated gel electrophoresis system comprising multiple, miniature gel channels, each 2cm long and just 1.5mm wide. As with polyacrylamide gels, the click gels are added into the channels as a solution, with the polymerization process taking just 10 minutes if the gels are heated to 70°C.

Fluorescent gel

As hoped, the click gel performed much better than the polyacrylamide gel, able to separate the DNA ladder into clear bands with significantly improved efficiency and selectivity and much less band broadening. In addition to being able to incorporate the fluorescent compounds, another reason for the click gel’s enhanced performance was that heating ensured a more uniform gel than exposure to UV light. This is because certain regions of the acrylamide solution often receive more illumination than others, producing a variation in the viscosity of the gel that can cause band broadening.

Next, the chemists compared their click gel with an agarose gel, which can also incorporate fluorescent compounds but isn’t very good separating small DNA strands. Again, they tested the gels on a DNA ladder extending from 100bp to 1500bp, finding that the click gel performed better, with greatly improved efficiency and resolution. The only downside was that the click gel took much longer to separate the DNA ladder (20 minutes rather than 90 seconds), but it just shows that you can’t rush quality.

Journal of Chromatography A, 2017, 1513, 226–234 : "Synthesis of hydrogel via click chemistry for DNA electrophoresis"

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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