If you haven’t read our last post, head here to find out what happens to the wasps when you send them to us. Spoiler: we extract their DNA!
But now we’ve got the DNA, what do we do with it?
We’re looking at a gene called COI (Cytochrome c oxidase I) which is used as a barcoding gene.
It’s kind of like how when you’re at the supermarket, all the barcodes on items are very similar, and in similar places on the boxes. They’re different enough, however, that you can scan them at the checkout machine and the computer knows what item you are buying.
The COI gene is found in all animals, and is generally similar enough amongst animals (or ‘conserved’ enough – it hasn’t changed too much over the course of evolution) that we can sequence the gene pretty reliably, but different enough that it is often unique for each species. Of course, it’s not always that simple!
To allow us to sequence and compare the COI gene from our different wasps, we first do a PCR (polymerase chain reaction). You might remember this from high school biology – but we’ll take you through it step by step.
DNA is a double stranded molecule shaped in a double helix.
In a PCR, we break apart the DNA strands (the green things in the diagram below) and use primers (the red things in the diagram below) to bind to the section of the DNA molecule where the COI gene is located. Then we allow free nucleotides (small pieces that make up the DNA – the dark blue things) to bind and extend our copy of the DNA strand – in this case, just the part of the DNA which is the COI gene. Then we repeat this many times to get many many copies of the COI gene – enough to sequence!
So what does this look like in the lab?
We work in the super clean PCR set up lab, and add our enzyme, some water, the free nucleotides and the primers to tubes. Then we add the extracted DNA from the wasps.
We run the PCR on these machines. We input a program which heats and cools the tubes over the cycles of PCR.
Once the PCR is done we need to know if it worked or not. For this we run a gel. The gel is made of a substance called agarose which is a liquid at higher temperatures. We pour it into the mould above, and as it cools it sets like jelly. The little white comb in the gel allows us to make wells or depressions, to put the PCR products in. Once it sets we add our PCR products to the wells in the gel and run an electrical current through. The smaller pieces of PCR product will move faster through the gel, and the larger pieces will move slower. This allows us to separate out the products in the tube and visually inspect what happened during the reaction!
Then we need to stain it! We put the gel into a stain which binds to any DNA (our PCR products).
We can only see the stain under UV light, so we put the gel into a machine with a strong UV light and a camera.
And hey presto! We can see our PCR products. The images below mean we got a good result! Each of those black smudges are in line with each other, which means we only made copies of one size (that’s good!). Also, we can see a black smudge on every lane, which means all the sample underwent the PCR reaction successfully. Now to sequence them!