An Extreme Solution – Polymerase Chain Reaction

Thermus aquaticus

                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.

REFERENCES

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, 5^th Edition.

An Extreme Problem

                 Let’s talk about DNA. 

                In my Central Dogma post (March 2011), I told you about DNA.  I’ve copied that paragraph and its figure (Figure 4.4) here: 

A molecule of DNA is a long string of As, Gs, Cs, and Ts, which are also called bases.  Much like a spread out charm bracelet, DNA is a long backbone (bracelet) with individual bases (charms) coming off the backbone.  Because DNA is a double helix, it is has another backbone and another set of bases on its other side.  If we know what one side says, then we automatically know what the other side says because of base pairing.  A is always paired with T and G is always paired with C.  Figure 4.4 gives you a nice diagram of a DNA molecule.

The bases of DNA are transcribed into RNA which is then translated into protein (just re-read the whole Central Dogma post if you think I’m speaking gibberish).  These proteins then fulfill all sorts of roles in our cells that keep us healthy and alive.

A gene, which is a string of bases that encodes one protein, is quite long.  If your protein is 100 amino acids long (small protein!) then the gene that encodes it must be at least 300 bases*.  That is at least 300 total As, Gs, Ts or Cs all linked together.  Large genes can have thousands of bases strung together.  The entire human genome is 3 billion bases!

How easy is it to string bases together?  How fast can cells replicate DNA?  How fast can scientists replicate DNA?  Can scientists just string together any order of bases in their labs and make new genes?  

Well… you’ll see that cells are really really good at replicating DNA, while scientists are mere shadows of the cells’ machinery.

Cells

Figure 26.1 shows you a long stretch of DNA comprised of two strands of bases: strand X and strand Y.  When it is time for DNA to replicate, several specialized proteins go to the DNA and pull strand X from strand Y.

A very important protein called DNA polymerase now binds to strand X.  Using strand X as a template and free bases floating around the cell, the polymerase makes an entirely new strand Y.  Remember that if you know the sequence of one DNA strand, then you must know the opposite strand because of base pairing.  A always matches with T; G always matches with C.  

Another polymerase will bind to strand Y and create a new strand X.

Now, we have two complete DNA molecules instead of just one.

DNA polymerase is extremely good at stringing together bases to make new DNA molecules.  

Polymerases can also move quite quickly.  The DNA polymerase from Pyrococcus furiosus (called Pfu Turbo) can link together ~ 1000 bases in about 2 minutes.  Some polymerases also come with proofreading ability: after they add a new base and move on to adding the next one, a small part of the protein trails behind and double checks that the last added base pairs properly with the other side of the DNA.  Pfu turbo has an error rate of 1 in 1.3 billion bases added.  Not too shabby!

Benchtop Chemistry

                Scientists wanted to be able to create DNA with sequences of their own choosing in the laboratory.  In theory it seems simple – link an A with a T, then link the T to a G and onwards until you’ve made your whole gene.

                In practice, it’s actually really difficult.  The system organic chemists have worked out requires nasty organic solvents and must be completely devoid of water.  We have lots of glass bottles that can only be opened in controlled areas and with needles poked inside to spray nitrogen on everything that will be involved in DNA synthesis.  It’s awkward and time consuming to link DNA bases on the benchtop.

                At most, scientists can link together ~ 40 bases of DNA before yields drop off rapidly. 

                The time it takes to link two bases together with this system is approximately 2 minutes.

                Compare our methods with our cell’s methods.  Clearly, scientists are losing.

Combining Cellular Machinery with Benchtop Chemistry

                Scientists understood how cells pulled apart DNA and replicated itself.  They really wanted to do that outside a cell, reproducibly, in large quantities, and take advantage of the proteins involved.  The proteins were better than them and they knew it.  No organic synthesis currently devised could compete with DNA polymerase.

                Unfortunately, there were two problems.

One: DNA polymerases can’t just start a new DNA strand; they need to add bases to an already short stretch of bases (~ 10 bases).  These short stretches are called primers.

Solution: Use Benchtop Chemistry to create short primers.

Two: DNA polymerase requires a template to work from.  DNA is double stranded.  It must be pulled apart before a polymerase can start adding bases.  The easiest way scientists knew to pull apart DNA was to heat it to very high temperatures.  All the polymerases they had available would fall apart at high temperatures because proteins are very sensitive to temperature.

Solution: Use a polymerase from a thermophilic bacterium.  The polymerase would be stable even at very high temperatures because the host bacteria loves high temperatures and it was designed to work in those environments.

           Now, scientists can mix together one DNA double helix, a DNA polymerase from a thermophilic bacterium, and free DNA bases.  Heat the DNA up to pull the strands apart and let the polyermase do its thing.

           Voila.  Scientists can accurately make longer stretches of DNA (upwards of ~ 10,000 bases) in large quantities.  

            The next post will discuss the classical polymerase used for replicating DNA in the laboratory: Taq polymerase.

* Because of the way genes work, the actual number of bases will be far more than 300 for a 100 amino acid protein.  I just used this as an example that people could understand.

Bases: A, G, C or T.  These bases are strung together to make a DNA molecule.

DNA polymerase: A specialized protein in our cells that can link bases together and make new DNA strands

Free bases: A, G, C or T that not yet incorporated into a DNA molecule

Pfu Turbo: DNA polymerase from Pyrococcus furiosus, sold by several companies including Stratagene

Primers: short stretches of DNA that a DNA polymerase can add bases to

REFERENCES

Lodish, et al. “Molecular Cell Biology.” (2004) WHFreeman Publishing, 5^th Edition.

Living at Extremes

                 My apologies for not getting a new post up sooner – life got a bit busy over the past two weeks.  However, I have put together a three part mini-series on some really interesting organisms called extremophiles.  We’re going to cover some general information on them and their extreme living conditions first then I’ll focus on one particular organism, called Thermus aquaticus, and how one protein from that little bacterium makes lab life much easier for post docs like me.

                So.  What is an extremophile?

                Literally, they are organisms that live/thrive/survive in extreme environments.  Of course, “extreme” is in the eye of the beholder.  In this case, the beholder is human so anything beyond our norms is considered extreme.  

                According to the article I read in Nature, Romans used the word “exter” to describe anything being on the outside.  They eventually wanted (or “needed” depending on your view of the Romans) a word that meant anything really really beyond normal, so they coined the word “extremus,” which is now the English word “extreme.”  I have already covered the root of “phile” (see Soap! post) but just to really hammer it home: “philos” is Greek for “lovers.”

                When I was in college, I learned only about two types of extremophiles: thermophiles and halophiles.  It appears that a whole world of extremophiles was known and that my deep chemistry education didn’t allow for more learning time on the subject.  Sad!

                I’m going to walk through a few of them, describe their environments, and discuss how they deal with their “extreme-ness.” Remember that all life uses the same basic molecules on Earth (amino acids, DNA, etc) and extreme conditions are harmful to them.  These organisms must use these same tools but take advantage of their known characteristics to allow survival at these extreme conditions.

Thermophiles: 

About: Some like it hot. Humans live in the range of 0°F/-17°C to 100°F/38°C (generally speaking) and are called mesophiles.  Thermophiles like it a smidge hotter (140°F/60°C – 176°F/80°C) and hyperthermophiles achieve maximum growth at over 176°F/80°C!  Yikes.  The archaea bacterium Pyrolobus fumarii enjoys a balmy 234°F/113°C.  

Where: Where on Earth can you find these ridiculous temperatures?  Hotsprings, geyers, and deep in the ocean at the hydrothermal vents.  Interestingly, many different life forms exist around those vents (examples: worms, shrimp, bacteria).  It’s rather popular despite its location and hot climate.

Problems: membrane fluidity, protein/DNA denaturation

Solutions: Adjusting composition of membranes to decrease fluidity; changing protein structures to allow for more heat stability; using mono- and divalent ions to stabilize their DNA at these high temperatures.

Psychrophiles

                About: Some like it cold.  Psychrophiles enjoy temperatures below 59°F/15°C, which I’m sure doesn’t sound so daunting until I tell you that Himalayan midge is quite content at 0°F/-18°C.  

                Where: Very cold places like sea ice, artic soils, or deep ocean (not near hydrothermal vents, obviously).

                Problems: membrane fluidity, ice crystals, protein function

                Solutions: adjusting composition of membranes to increase membrane fluidity; rigidifying protein structure; proteins involved in keeping water from freezing

Halophiles

                About: Some like it salty!  Halophiles live in very very salty conditions. 

                Where: Salt flats, natural lakes and deep sea hypersaline basins

                Problem: Retaining water.   

               A cell is separated from its environment by a semi-permeable membrane (meaning things can pass across the membrane if they meet the right requirements).  If you make the area outside the membrane very salty, you will force water inside the cell to flow outside.  Why?  Science demands that the concentration of salt be the same on both sides of the semi-permeable membrane.  The salt concentration outside is very high and the salt concentration inside the cell is lower.  The easiest way to increase the salt concentration inside a cell is to remove water.  Cells need water to survive so having it all flow out leads to death.  (This is why you die if you drink salt water, by the way.)

                Solution:  The organisms utilize different strategies to sequester water inside their membranes so it is unavailable to flow out.

Alkaliphile

                About: Humans maintain a pH inside their cells of ~ 7 and most biological processes occur around this range.  However, alkaliphiles enjoy the pH much higher (> 9).   

                pH is simply a measure of how many protons are around.  A lot of protons?  Low pH.  Only a few protons around?  High pH.

                Where: soda lakes or drying ponds

                Problems: Protons are required for a cell to make energy.  At high pH, protons are scarce.

                Solutions: The organism tries to maintain pH of 7 inside their cells by bringing in as many protons as possible.  The organisms also have unusual permeability properties to their membranes.

Acidophiles

                About: Opposite of alkaphiles, acidophiles enjoy very low pH (< 3 or so).  Their environments have a lot of protons floating around.  Cyanidium caldarium (red alga) enjoys a pH of 0.5 while Dunaliella acidphila (greena alga) would prefer 0.  Helicopbacter pylori can live in our stomachs (pH ~2) and can cause ulcers.

                Where: sulphuric pools, geyers, hydrothermal vents, acid mine drainage, our own stomachs

                Problems: protein denaturation

                Solutions: Organisms have a lot of proton pumps to remove protons from their cells and bring the pH up inside their cells.

                These are just some of the few.  Other organisms exist that can live at incredibly high pressures, almost no water (or gravity!), high radiation or chemical extremes.  Some can live in environments that are combination of these characteristics; these are called polyextremophiles.

                Of course, this leads to two very interesting concepts: 1. Life in space, and 2. Usefulness of these organisms to the masses. 

 Learning that these extreme environments do not preclude life leads to some interesting questions about life on other planets.  Places that we previously concluded were inhospitable to life may have it after all.  Keep your eyes open!

As always, scientists (and business owners) are looking for new, cheaper or novel ways to make their products.  What if we could find proteins directly involved in keeping psychrophiles running?  What if we could purify them and apply them to frozen human organs?  Currently, we can't reliably freeze human issue (or bodies.  Want to revisit the Absolute Zero post?) due to ice crystal formation.  However, we might be able to exploit psychrophile adaption methods for our own gains.

One area that we have been able to use extremophile adaptations is the laboratory. Most scientists have used a little enzyme called Taq Polymerase. It is an enzyme responsible for linking nucleotides together into DNA.  It was purified from Thermus aquaticus and is currently sold to post docs like me.

Before we get into why this is so interesting, we first need to understand DNA. 

Extremophile: an organism that lives in extreme environments

Polyextremophiles: an organism that lives in an environment that is extreme is more than one way.

REFERENCES

Rothschild and Mancinelli. "Life in Extreme Environments." Nature (2001) 409, pgs 1091 - 1101.

More info about hydrothermal vents:

http://www.onr.navy.mil/focus/ocean/habitats/vents1.htm