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, 5th Edition.
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