All posts by Erinn Fagan-Jeffries

What do we do with the DNA?

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.

Box, Cola Dose, Cola, Drink, Brand, Erfrischungsgetränk
The barcode on the coke can is unique – when you scan it at the register, the computer knows it’s a can of coke! Image: Public Domain WikiCommons

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.

File:DNA simple2.svg
Image: Public Domain WikiCommons

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!

File:Polymerase chain reaction.svg
Image: CC-BY-SA Enzoklop WikiCommons

So what does this look like in the lab?

IMG_2278We 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.

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We run the PCR on these machines. We input a program which heats and cools the tubes over the cycles of PCR.

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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!

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Then we need to stain it! We put the gel into a stain which binds to any DNA (our PCR products).

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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.

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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!

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What happens to the wasps you send us?

The first thing we do when we receive packages of parasitoids is to look at them under a microscope. As much as we can, we then work out what they are using identification keys.

Here is an example of a simple identification key to insects orders.

A lot of parasitoids, particularly the subfamily of wasps we are looking at (the microgastrines) are not yet described by taxonomists. Even for those that are, we’re definitely not experts in every type of wasp! This means that whilst we might be able to work out which family and genus of wasps the parasitoid belongs to, we might not know what species it is. That’s where the DNA comes in!

Getting the DNA out of the wasp

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The first step to doing a DNA extraction is to remove the part of the wasp you want to get DNA out of! Often we will just use a couple of legs, so that the rest of the wasp stays intact and we can look at it later.

Once the legs are removed we put them in a small tube and take them to the DNA extraction lab.

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We always wear gloves during a DNA extraction, as we don’t want to get any of our skin cells (which are full of OUR DNA) into the tubes and contaminate them.

In the DNA extraction lab, we add something called a lysis buffer, which breaks down the cell walls of the wasp so that we can get to the DNA. We also add an enzyme which starts digesting (breaking down) the proteins, which would otherwise get in the way.

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Then we put all the tubes on a heat block, which keeps them nice and warm (55°C) overnight so the lysis and enzyme can do their stuff!

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The next day we remove the proteins and any other material that is in our tube, purifying the DNA. We add isopropanol (a type of alcohol) and glycogen. The DNA and the glycogen are insoluble in water, so will separate out, whilst any material left in the tube that is soluble in water will stay in solution. The tubes stay in the freezer overnight, and then we spin them in a centrifuge to make the DNA clump into a tiny ball at the bottom of the tube. The centrifuge (below) spins the tubes really fast, making heavier things (like the DNA) go to the bottom of the tubes.

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The last step is to do a wash with ethanol (another type of alcohol) and then add a buffer that the DNA will stay stable in for our future work! Easy as that, we’ve got purified wasp DNA ready to use.

Want to do your own DNA extraction? This is a good version of a simple extraction to use on your own DNA at home!