My friends and family often wonder what it is I actually do everyday. Lab work is very much a black box for people who have never been inside a lab or been privy to a friend’s lab tales.
How is it that we’re able to take an animal off the seafloor and eventually end up with genetic sequences that help us understand how that animal evolved and how it’s related to other animals in other ocean habitats?
I’m going to attempt to give you a bit of insight into how gene sequencing works and why it’s important. Hopefully you’ll find this interesting. I’ve inserted some hypertext links to wikipedia sites if you want to read more about a specific term or concept.
If you’ve read my previous entries, you know that I was recently on a research cruise to the Mariana Trench. On that cruise, we pulled up several biological specimens from the bottom of the ocean using HROV Nereus.
When you get an animal on the research vessel’s deck, you want to preserve or freeze it as fast as possible. Sometimes, if it’s in a shell or a tube (for example a clam or a tube worm), we will dissect it out of it’s shell/tube in order to get the most amount of tissue exposed. This helps with preservation in alcohol because it means that more alcohol can reach more tissue right away, saturating all the cells and preserving them. We also want to expose the most tissue for frozen samples because that way we have more to work with when we take a piece off for DNA extraction.
Here's a picture of me dissecting out a specimen on the cruise:
Often times, the animals that scientists collect while at sea don’t immediately get back to the lab. In the case of the Nereus samples, they stayed on the ship until it came into port in Hawaii and then were sent over-night in a cooler to Woods Hole.
Building Blocks (ATCG)
For those of you who haven’t thought about DNA since high school biology, I’ll give a very brief description how it is structured. Basically, DNA consists of two long chains of genetic building blocks called nucleotides lined up next to each other. There are 4 kinds of nucleotides in DNA: adenine, thymine, cytosine, and guanine, also known as A,T,C, and G. Each one is a little different from the others and they form specific pairs that we call base pairs. Meaning that A only likes being across from T and C only likes being across from G.
In all of your cells, DNA lives in the nucleus, a well-protected compartment that keeps all 3 billion of your base pairs organized and accessible to the cellular machinery!!
An animal cell (nucleus labeled), from a website I found on google images:
There are many different methods used to extract DNA. They all have a few things in common, however. First of all, you have to essentially destroy the cells in the sample in order to free the DNA from the cell’s nucleus. As you might imagine, DNA is pretty well protected by the cell. Using chemicals and enzymes, usually mixed with heat and some physical agitation, the first step of any extraction is to free the DNA by digesting the cell around it. You have to disrupt the cell membrane and try to digest proteins in the cell.
What does that mean for me? It means that my first step in a reaction is to put a very small piece of the animal’s tissue in a tube with a specific set of reagents that break up cells. Then, I leave that tube in a warm water bath for a while (usually overnight) to help the reaction go faster.
These are the water baths in our lab:
The next day, I go through a number of steps to isolate the DNA chemically from the other fragments of the exploded cell. Since DNA has a specific structure and is made of certain molecules, it has specific chemical properties that we can exploit to isolate it from other molecules. Each of the different extraction methods exploits these properties in different ways but in the end, you should end up with somewhat pure DNA.
Once you have separated the DNA from other cell parts, you put it through a series of reactions that makes many copies of one specific gene. Essentially, you selectively copy the gene you want to study by using enzymes that link nucleotides together in the same way that enzymes copy DNA inside a live cell. This process is called a polymerase chain reaction, or PCR.
After we’ve made many copies of the one gene we want, we run a sequencing reaction. A sequencing reaction is somewhat similar to PCR with a really amazing twist. In PCR, you add a mixture of nucleotide bases for the enzymes to use as building blocks. In a sequencing reaction, some of those nucleotides force the termination of the DNA strand because they are chemically different from normal nucleotides and don’t allow other nucleotides to latch onto them. These special building blocks are also labeled, or tagged with molecules that glow specific colors under certain conditions.
Since you are essentially making copies of the many PCR-formed copies of your desired gene, and you are using these special nucleotides that stop, you theoretically end up with many different lengths of DNA strands—from one nucleotide to the whole length of the gene in question. (Meaning, you’ll have a mix of the following: A, AT, ATT, ATTG, ATTGC, ATTGCC, ATTTGCC, etc.) Statistically, the nucleotide mix is meant to have enough of each type of nucleotide for this to work.
There’s a really incredible machine that can read these glowing sequences. It goes through and says, “This fragment is 5 nucleotides long and the last one glows red, so we know that the fifth nucleotide is a thymine.” A computer listens to the machine and stores this info. We get the sequence back and can then use software to read it to (first) see if everything worked, and (second) use the sequence to find out interesting things about the animal.
The data comes back to us not only in text files, but also with what’s called a chromatogram that shows how strong each color signal was and if there were conflicting color signals.
A chromatogram looks like this (the top row of this looks like a pretty good sequence; I pulled this off a google images):
What we do with the sequence could take up another really long blog post. So I will refrain from telling you all about that until a later date.
Until next time,