Capillary Electrophoresis

We’ve done our amplification step, and now we have all our targeted area, all amplified in 1,000 and million copies of DNA into our tiny tubes. But now we need a method to detect all the targeted amplicon we have amplified, and to do so, we’re using our capillary electrophoresis instrument. It works through exploiting one of the main characteristics

of DNA: the fact that the double strand of DNA is negatively charged. So, we’ll run a power supply through the positive charge to the positive poles.

So, the main reagents we’re going to use the steps are formamide, size standard and allelic ladder. Formamide and size standard are already mixed, and I already put them in the plate. So what we need to do now is add in one microlitre of our sample into the plate and one microlitre of allelic ladder as well. So, the size standard acts as a standard of the sizing of the fragments so the instrument will know how big is the fragment which is passing through the capillary, and you can compare it to how big is the fragment passing through the capillary from the sample well.

Whereas the allelic ladder is actually a conjunction of all the known STR markers in, at least, of every single loci, and it will help when the software is analysing the sample to assign a numerical value to the peaks that will be detected. So let’s add that now - the sample.

We’re going to use a Mohr pipette, and one microlitre of homogenised allelic ladder decause we want every fragment to be equally present in this mix.

Great. So we’re going to cover with a scepter and place this on 100 degrees. This step is particularly important because after PCR amplification, the DNA fragmented is now double-stranded again, and with this step, we want to separate the DNA into single strands in order to pass it through the capillary. Oh, I see. So, this step normally takes about five minutes, and then we’re going to need to cool down.

OK, so now we’ve waited five minutes at 100 degrees, and the DNA is now single-stranded. So we’re going to place it into a cooling block so the DNA will be trapped in the formamide and maintain the single-strand feature, and it will be ready to be run through the capillary. So, in the meantime while we’re waiting for this to happen, we can pull the tray in. So please have a look and see how the capillary actually works.

This is the bay where the plate will be loaded.

This is the polymer - the polymer is basically a net that runs through the capillary, and the smaller is the fragment that we are analysing, the quicker is the action of the fragment to go through the capillary because of the net. So, the bigger the amplicon, the more difficult it is to go through the net, and so it will take longer time. So this is how the capillary is separating not only by size but also separating in time. So this is the capillary, and it will run through here, and here it will be hit by a laser, and the laser will develop a colour, because if you remember, our polymers are fluorescently labelled.

The colour will be detected by the software afterwards. So we’re now ready to load.

OK.

So we’re ready. We’re going to place the plate and press start. So, everything is done. So DNA profiling processing, we ran

through everything: extraction, amplification and detection. So now, we just have to wait for the results. Now, let’s pass it over to Dr David Ballard to see how our works look like from today. OK, thank you.