DNA replication is carried out spontaneously in living organisms (in vivo).
DNA, of course, replicates. Why? It’s a pretty crucial element in the reproduction of living things. For example, a bacterium replicates by splitting itself into 2 (binary fission). The DNA must stay intact and be copied with a high degree of accuracy in order for the two newly formed bacteria to develop and function as their parent – adequately. In multi-cellular organisms such as ourselves, DNA replication occurs as a prelude to cell division.
For such a complex molecule, past scientists have had a challenging time working out the precise mechanism by which DNA replicates. Three hypotheses were made: (for this purpose imagine one DNA molecule)
1. The DNA molecule replicates by providing itself as a template for a brand new shiny DNA molecule, and then remaining its own intact DNA molecule. This is called the conservative replication model.
2. The DNA molecule replicates by providing itself as a template and being modified itself throughout, resulting in 2 new DNA molecules with patches of the old parent DNA molecule combined with patches of brand new material. This is called the dispersive replication model.
3. The DNA molecule replicates by providing each of its strands as a template for 2 new DNA molecules, each having one entire new strand, and one entire old strand from the parent DNA molecule. This is called the semi-conservative replication model.
Here’s a visual aid for those who found the above descriptions gibberish:
How does one go about working out which one of these models is the correct one? Well, in the ’50s these two chaps by the names of Meselson and Stahl cracked the riddle by carrying out a classic experiment which the examiners are in love with (so learn it well). Such complicated affairs can only be properly depicted by a lovely video. Videos always give the impression that what you are watching, surely, must be a piece of entertainment rather than advanced biology.
Now that we’re all clear on the replication model of DNA, let’s delve deeper into the details of the process. These are the key steps involved in the semi-conservative replication of DNA:
1. The enzyme DNA helicase unwinds the double helix, causing the hydrogen bonds between the two polynucleotide strands to break.
2. DNA-binding proteins maintain the 2 strands separate during replication.
3. Enzymes called primases attach primers to the exposed strand. Primers are a few nucleotides long and constitute the site where DNA polymerase starts its action.
4. DNA polymerase binds to the aforementioned primer and begins catalysing the reaction between free nucleotides (new) and DNA-bound nucleotides (old). Of course this complies with the principle of complemetarity i.e. A-T, C-G. The direction of nucleotide addition is from the 5′ end towards the 3′ end.
5. Because one strand is replicated continuously while the complementary strand is replicated backwards, and hence in fragments (Okazaki fragments) rather than continuously, the resulting DNA fragments must be connected together to form the new strand. New phosphodiester bonds between the sugar-phosphate backbone of DNA are catalysed by the enzyme DNA ligase.
Here is a good video animation of this process.
DNA can also be replicated in the lab (in vitro) by isolating the individual components required, such as enzymes, and adding a template DNA to the mix.
If we have obtained a DNA sample or a few, what next? Well, nothing much can be done with that. We must obtain exponentially more DNA to use for any purpose. And it all of course must be identical. We must essentially clone our DNA. Considered the very staple of molecular biology, this technique for multiplying DNA many-fold was invented by a chap Kary Mullis who believes in astrology.
The DNA template to be amplified can be extracted from a field sample (a leaf, human saliva, cultured microorganisms, etc.) or synthesised chemically, on order.
Essentially the DNA is denatured so the 2 strands break apart, short complementary bits called primers attach to the strands, the enzyme DNA polymerase binds to the primers and initiates the assembly of a new DNA strand, and finally the process is repeated many times over in a chain reaction. This is the polymerase chain reaction, PCR.
Soon enough, the few bits of DNA become thousands, and hundreds of thousands, and millions…
The components of PCR can fit in a very small tube which is placed in a specialised thermocycler or water bath in order to expose it to these fluctuating temperatures. Thermocyclers can be programmed to run automatically on a cycle along the lines of (degrees Celsius) 90-60-70 each for a few minutes, repeated many times over e.g. 30 times. Overall, this can take a few hours to complete.
The fluctuations in temperature correspond to each step in PCR. The highest temperature is required to separate the strands. The lower, annealing temperature bring the strands closer again, in order to bind the primers required to kick-start replication by DNA polymerase, while the temperature lower than the denaturing step, but higher than the annealing step is required for the addition of nucleotides by the polymerase – extending.
These temperatures are well above most physiological conditions where enzymes like polymerase would be functional, so special polymerases are used in PCR which are heat-resistant. They were isolated from microorganisms found living in hot springs and such extreme environments.
Other ingredients of PCR include the nucleotides themselves (free and ready to be added to new DNA strands by polymerase), other optimising agents such as magnesium ions for the DNA polymerase, and water.
Controls and applications
Positive and negative controls are used for PCR. Controls ensure that the outcome of the experiment is what it seems to be.
Positive controls give a reference point for what the result would look like if it worked, while negative controls give a reference point for what the result would look like if it didn’t work.
A positive control for PCR might be a PCR reaction identical to the one we are running as an experiment, but instead of the test template DNA we add a different template DNA that we know will definitely work based on previous data. If the experiment fails, but the positive control works, we can be sure that the PCR reaction was correct but there was an issue with the test template DNA.
A negative control for PCR requires a little less sophistication, and might involve using the same PCR reaction while omitting any template DNA at all. If we seem to get something that looks like it worked in our experiment using our template DNA, but it looks the same as the negative control, then we can be sure that it actually hasn’t worked, and the result is because of another reason e.g. contamination, background signal, PCR ingredients themselves, etc.
Visualising DNA with gel electrophoresis
A common method of visualising differences is gel electrophoresis which involves loading small volumes of samples on a gel and running a current across it in order to separate the samples by size.
Since the gel has a microscopic matrix inside that provides resistance against sample movement through it, the larger molecules move more slowly while the smaller fragments can move more quickly.
The positive charge is at the bottom of the tank, while the samples are loaded at the top. This way, they will move downwards towards the bottom of the gel because they have a negative charge as molecules. The current is run across the gel for around 30-60 minutes (ensuring the samples don’t run too long and hence run off the gel into the buffer solution! if that happens they are lost) after which the sample’s progression on the gel can be visualised by using a stain solution or pre-existing coloured label visible under UV light.
Any 2 given people share 99.9% of their DNA code. But the differences present in the remaining 0.01% of it are enough to enable reliable identification, with the exception of monozygotic twins. The DNA containing this is called variable number tandem repeats (VNTRs) because they are just sequences of DNA repeated many times.
Aside from genes, or coding DNA, there are non-coding regions which repeat themselves many times over in each individual, with some sequences contained within varying. This variability is less in closely related individuals. This is where the usefulness of genetic fingerprinting comes in. This covers medicine, criminology and biodiversity conservation among other things.
1. The sample DNA undergoes PCR then cleavage at multiple sites with restriction endonucleases
2. The resulting many small fragments are tagged using a radioactive molecule
3. They’re separated using gel electrophoresis and viewed using a developed photographic film
The bands exposed then undergo simple visual analysis by matching up the template DNA with other DNA that could be similar more or less, depending on situation. Above, the DNA found at a crime scene is compared with that of 3 suspects. The bands of suspect 2 are perfectly aligned with the crime scene DNA.
In the case of paternity tests, the child’s DNA fragments do not completely match their father’s, because some will be from the mother. Here, only the remaining fragments (the ones not from the mother) are matched up against potential fathers.
Mary, the mother, and the child share the first fragment; so looking at the remaining fragments of the child, Larry is the father as they share 3/3 fragments. Bob and the child only share 1/3.