In 1969, the same year as man was landing on the moon, scientists were also exploring our universe on a much smaller scale. The thermophilic bacteria Thermus aquaticus was characterized in an article published by the Journal of Bacteriology.
Let’s look at a few pictures of it, yes?
Typically, the bacteria form long filaments, but their size and overall structure vary depending on the temperature. In older cultures, it is common for the bacteria to form spherical bodies (Figure 27.1). Not being a microbiologist, I’ll let you read the reference if you are interested in how it reproduces, grows, forms spores, etc.
T. aquaticus can be found in many thermophilic environments. The bacteria enjoy a balmy 70 - 75°C (158 - 167°F). The authors studied several hot springs located in Yellowstone National Park in addition to Pacheteau’s Calistoga in Hot Springs, California. Several different strains of the bacteria could be isolated from these places. The authors also investigated hot water from a building at Indiana University. They found T. aquaticus living quite contentedly there, as well. Fascinating.
At the end of my last post on this series, I told you that scientists use the DNA polymerase from T. aquaticus to make DNA in the laboratory. We call this protein Taq (short for T. aquaticus – we’re geniuses with naming). I went through it very quickly so I’m going to explain it better now. Yay for you.
It all starts with a short stretch of DNA. Don’t worry about where you got it, just assume you have it. This short stretch of DNA probably encodes a protein that your laboratory is quite interested in. You want more of that short stretch of DNA. This is where it all starts. You have a little, but you need more. (“No sir, I have no experience but I’m a big fan of money. I like it; I use it; I have a little. I keep it in a jar on top of my refrigerator. I’d like to put more in that jar. That’s where you come in.” –Robbie Hart)
I told you that using organic chemistry to recreate this short stretch of DNA is cumbersome, time consuming, and sometimes plain impossible (see An Extreme Problem post). Cells, whether they come from humans or bacteria, have a built-in machine to easily make DNA called DNA polymerase. It’s a protein that can take bases of DNA that are just floating around (free bases) and link them together to create a new DNA molecule. DNA polymerases are fast, accurate, and basically wonderful.
Figure 26.1 (of the An Extreme Problem post) shows you the basic way DNA is replicated in a laboratory. I’m now going to go through that in more detail. This laboratory method is called the Polymerase Chain Reaction (or PCR).
Step One: Mix together short stretch of DNA that you want more of, free bases (As, Gs, Ts and Cs), Taq, and primers. These pieces will be discussed in more detail as we go along.
Step Two: In order for the DNA to replicate, the two strands must be pulled apart. The easiest way to pull two strands apart in a laboratory setting is to heat it up to ~ 95°C (203°F). This heat doesn’t hurt the DNA molecule aside from pulling the two strands apart; the free DNA bases and Taq don’t mind the extreme heat.
Step Three: Cool the reaction down slightly (~50 - 60°C) so that primers can bind to the DNA. Primers are short stretches of DNA that are complementary (base pair properly) to the beginning of each strand of DNA. We need primers because DNA polymerase cannot start a DNA strand on its own, it must add bases to an already existing strand. Primers are made using organic chemistry because they are so short. We can make these primers ourselves or order them from a company (IDT being the best!).
Step Four: Increase the temperature slightly to the optimal temperature for Taq to work (72°C). The polymerase will bind the DNA and use the free bases to create a new strand of DNA.
Fantastic! You’ve successfully gone from one molecule of DNA to two.
What if you want more? What if you heat up the reaction again to 90°C?
Well, your two DNA molecules will now come apart. Once you allow primers to bind and Taq to do its thing, you’ll have gone from two DNA molecules to four DNA molecules.
What if you do it again? You’ll go from four DNA molecules to eight DNA molecules.
See how you can quickly end up with many many many copies of the same DNA molecule? It only takes a short time, too! One run of Steps 2 – 4 takes ~ 2 minutes.
Typically, when scientists perform this technique, they mix the DNA, primers, Taq and free bases in a plastic tube. This tube is then placed in a machine called a thermocycler. We design a program for the thermocycler to heat up and cool down the tube to very specific temperatures. For example, we tell the machine to heat the tube up to 95°C for 30 seconds, then to cool the tube to ~ 50 - 60°C for 30 seconds, then to warm the tube up to 72°C for ~ 1 min. Given the steps written above, you should now know exactly what is going on at each temperature.
What would happen if we used human DNA polymerase instead of Taq?
Well… humans live optimally at ~ 20°C. Our proteins are designed to live within certain temperature ranges. If we heat up our own DNA polymerase to 95°C, then we will destroy it. It simply isn’t stable at those temperatures. However, Taq was designed to work at much higher temperatures because T. aquaticus thrives in the heat. Without the discovery of thermophilic bacteria, we would be unable to use the polymerase chain reaction as outlined above.
Taq is not the only thermophilic DNA polymerase on the market (Taq is sold by Promega, Roche, and Invitrogen). The other popular choice is Pfu Turbo which comes from another thermophilic bacterium and is sold by Stragene, Fermentas and Invitrogen. They both have their pros and cons so scientists pick accordingly.
Polymerase Chain Reaction – a series of steps performed in the laboratory that will take one molecule of DNA and replicate it two; these two DNA molecules are then subjected to the same series of steps to create four DNA molecules. This is repeated many times to create many copies of DNA.
Complementary – One strand of DNA base pairs with the other strand to create a DNA molecule. These two strands are said to be complementary because they base pair correctly with each other.
Thermocycler – a machine used in laboratories to heat up and cool down tubes.
Brock and Freeze. “Thermus aquaticus gen. n. and sp. N., a Non-sporulating Extreme Thermophile.” J. of Bacteriology (1969) 98(1), pgs 289 – 297.
Coraci, Frank. (1998) “The Wedding Singer.”
Lodish, et al. “Molecular Cell Biology.” (2004) WHFreeman Publishing, 5th Edition.