Wednesday, September 25, 2013

Research: The Lab

So you've seen where I it's time to learn more about what it is that I actually do.

As mentioned in my last blog, I collect samples in the Maryland and Virginia coastal bays. What that means, is that I tow a plankton net and collect a sediment sample at each site, as well as getting the environmental data.

Drying the ethanol out of the DNA samples.
Alcohol breaks down DNA, hence why you can sterilize
stuff with it.
Like open wounds.
Those plankton and sediment samples are taken back to the lab. The sediment samples are stuck in the freezer...indefinitely at this point. I am more concerned with the plankton samples...and I don't want to work with dirt.

So the plankton samples come back to the lab and I purify them so that all that remains is the DNA from the organisms that were present in the original sample. This process entails a lysis step, which means the cells get broken up, so that access to the DNA can be granted. It also involves an RNase step and a Proteinase K step. These steps are where the RNA and proteins are broken down and ultimately removed from the final product. Thus, I end the multi-hour process with 200 micro-Liters of clear liquid, which hopefully has some DNA in it. For those of you who don't know, a micro-Liter is 0.000001 Liter.

Pipette tips; pretty much the least cool thing ever.
That's why you need instagram filters.
Now I've got a tube with some clear liquid in it....which I hope has some DNA. Even better, if it has the DNA of the specific parasite I work with. So how do I figure out if it's in there?

That's where the real science comes in!

We use PCR, which stands for Polymerase Chain Reaction, to amplify the DNA in our sample. And, depending on what we put in to the reaction, we can amplify specific strands of DNA. So I can amplify only the DNA of the organism I work with, or I can amplify the DNA of any organisms that belong to a broader group. A group might be anything from all vertebrates (Phylum: Chordata) to just mammals (Class: Mammalia).

No, these are not my hands.
The way PCR works is that we create a master mix that has all the essential building blocks of DNA. That means the nucleic acids and some salts. It also means the taq polymerase that will do the actual building of new DNA and the primers that identify the region of DNA that is to be amplified. Each sample is put into a small tube with some of this master mix, and placed inside a thermocycler which heats and cools the DNA so that it can replicate.

If you remember basic high school biology, DNA is double stranded and coiled into a helix. It's made up of 4 nucleic acids, Adenine, Guanine, Cytosine and Thymine. A binds to T and G binds to C. Thus, when you split a strand of DNA, you can rebuild the other side following the base pairing. That means you started with one strand, split it and ended up with two strands. The next reaction will leave you with 4 strands and the one after that will leave you with 8 strands. This means we are exponentially amplifying the original DNA.

So why are we trying to amplify the DNA? Well, we are working with such small quantities, that we need a lot in order to actually visualize the DNA. So basically, we don't know if the DNA we want is present in our sample, but if it is, we want to increase how much is there, so we can actually detect it.

This is an issue because with the type of PCR we do, called end point PCR, the ultimate verification is pretty simple and easy to mess up.

This is what a gel looks like when you get desperate
 and take a picture of it with your phone.
Actually, maybe 'ethidium bromide' should be an
instagram filter..
When our amplification is done, we load the DNA into a gel. The gel is made with agarose (think know, like gelatin but only not for eating) and a small amount of ethidium bromide, which fluoresces under UV light.

Once the DNA is loaded in the gel, we run electrical current through it. It kind of looks like we are hooking the gel up to a car battery and since DNA has a negative charge, it move from the negative end to the positive end. As the DNA moves through the gel, is picks up the ethidium bromide AND separates out into different chunks; because the smaller pieces of DNA move faster than the bigger pieces. We make sure to put in a DNA ladder, which breaks out into known pieces of DNA, so we know approximately how big our unknown pieces are, and we also put in something that we know has the DNA we want to look at, so we have a reference for samples that are positive.

So we go through all this work to look at a chunk of jello and decide, 'Is the DNA I am looking for here, Y/N?'
The positive is positive, the negative is negative and Site 10 is also positive...
So I do all this work and all I get is a yes or no answer. We don't get to know how much DNA is present or what form it is in (you know, like a living organism or a dead organism). So this information can only take you so far....but at least we can monitor when and where we see our parasite.

While all this is interesting, it's just basic now I have to do something to make this project a little more novel.

And that's what I'll talk about in my next blog within this series...