This week I have been working on an experiment I have been learning called the Comet assay and so I wanted to tell you all a little bit about how it works. Like a lot of the experiments I’ve been learning lately, its one that allows us to assess the damage of DNA in a cell and is used to see how damaging radiation and chemical exposure can be to our tissues.
Before you start, the cells must be grown and then exposed to the radiation or chemical agent you want to test. The cells are then mixed with a thick substance called agarose which is a lot like jelly and sets into a gel when cool. The cell/agarose mix is quickly transferred onto a microscope slide and, as the agarose gel cools down, the cells become fixed within the gel pores.
The microscope slides are then submerged in a solution called lysis buffer, which causes the cell’s membrane to become disrupted and dissolve away, along with the proteins and other unwanted bits of the cell. Once the cells are completely ‘lysed’, the only thing that remains in the cavity where the cell once existed are the strands of DNA.
Now that the DNA has been isolated from the rest of the cell, the DNA must be separated further to assess how much damage there is. This can be done using a lab technique known as electrophoresis which separates charged molecules of different sizes using an electrical current. DNA is negatively charged and so when a negative charge is applied to it, the DNA is repelled and travels towards the positive electrode. Bigger DNA molecules take longer to migrate through the pores of the gel than smaller ones and so the fragments of DNA get easily separated by size. When a negative electrical charge is applied to the DNA on the microscope slides, any damaged DNA becomes relaxed and moves out of the cavity left behind by the cell; any undamaged DNA is too big and so cannot move out of the cavity.
When the DNA is finally stained, the microscope slide can be looked at under the microscope. The DNA that can be seen looks a lot like a comet shooting through the sky and so that’s where the name for the experiment comes from. If you calculate the proportion of DNA in the comet head (undamaged DNA) versus the DNA in the comet tail (damaged DNA), then you can get a direct indication of how much damage the radiation or chemical did to the cell.
The whole process is pretty straight forward and only takes a couple of days but does have some difficulties – the entire experiment should be carried out in the dark to prevent the DNA from becoming damaged from the light and interfering with your results. The first couple of times I tried the experiment I had to work in the light because learning new experiments are tricky enough, without adding to that the fact that you won’t be able to see majority of what you are doing! But once I got used to the techniques I didn’t find it too strenuous to switch to working in the dark. And luckily, we have been able to collect a good amount of data from it already – let’s hope for some interesting results!