Everyone knows what DNA is–the genetic code, the building block of life, what catches the criminals in CSI. But do you know how scientists harness the awesome power of DNA in their everyday research?
Marine science (like any other science these days) is becoming more and more reliant on genetic information. DNA can tell us a fascinating story—a book that has been written for millions of years, but whose existence has only recently been revealed. DNA is my Silly Putty; in the lab, I can do so many things with one lump of stuff.
In this new series of blog posts, I’ll be giving you the down low on biotech–the genetic methods we’re using today in the lab (and what we’ll be using in the future). The hook? I’ll be describing these highly sophisticated, cutting edge technologies in the most low-tech, ghetto way possible: drawings I make in Paint.
Part I: The polymerase chain reaction
We’re going to start with the Godfather of all lab work: The Polymerase Chain Reaction, a.k.a. PCR. The genome is the cookbook, but a lot of times you’re only interested in one recipe. The recipe (gene) you’re looking for depends on the question you’re asking. If I want to find out where a species might fit on the grand Tree of Life, I will focus on a slowly evolving, conserved gene like the one that encodes the small subunit of the ribosome (ribosomes are pretty integral to cell function, and if you screw up their recipe then your cell is basically F&%*D). If I’m more interested in describing population differences within a species, I’d want the sequence of a rapidly evolving gene such as the mitochondrial gene encoding the protein Cytochrome C Oxidase subunit 1 (the infamous “barcode of life” gene).
PCR is simple–all you’re doing is making photocopies of your target gene. But first, you need to break open the cell and nucleus in order to free the coiled DNA:
Typically, I pick nematodes out of deep-sea mud (using an eyelash mounted on a paintbrush handle) and put them into a liquid buffer containing Proteinase K; this enzyme eats away at the cellular structure, so after two hours on the heat block you’re left with free-floating DNA. You’d pretty much do this same process, with a few tweaks, regardless of the species you’re working on—tissue from fish, mice or even humans.
Next, we mix together tiny volumes of different liquids from a kit, throw in a little of your extracted DNA, and put the tubes on a machine where the magic happens:
PCR is all about the temperature cycles—you could easily replicate DNA at home, on your stove, if you have all the ingredients. The liquids we put into our reaction are: free-floating nucleotides (Adenine, Cytosine, Guanine and Thymine, used to build new DNA), the Taq polymerase enzyme (the workhorse that builds the new DNA strands), buffer (makes the enzyme happy and functional), and primers (specific, complementary sequences of DNA that stick to your target gene when the DNA is heated and unzipped).
Step 1 is called denaturation, where we heat up the DNA to a balmy 95°C, causing the double helix to unwind and separate. Step 2 is called annealing, where we cool the reaction way down and let the primers stick to both ends of the target gene (anneling temperature can be anywhere from 45-65°C, depending on the PCR recipe and primers you’re using). The final step is called extension, where we heat up the DNA to 72°C; this is the optimal temperature for the Taq polymerase to do its thang, adding nucleotides to the end of the primers and extending out our DNA copy over the entire length of the gene.
The coolest thing about PCR is the magical Taq Polymerase—nowadays, researchers just order this enzyme off the internet, but Taq was originally was discovered in Thermophilis aquaticus bacteria that live in hot springs and hydrothermal vents. This enzyme is totally our biotch (we can cook the $&%! out of it, and it STILL works), and the reason why PCR has become so ubiquitous and versitle in modern genetic research.
The second coolest thing about PCR is the primers. I can just call up a company and say “Yo, make me a synthetic strand of DNA that reads ATCTGCGGTGGG”. Two days later, it arrives in the mail. Isn’t technology awesome??
And finally, this wouldn’t be a ghetto guide to genetics without a ghetto PCR rap: