During replication of a DNA double helix the strands are unwound (separated) by a helicase and the single strands are used as templates by DNA polymerase III for the synthesis of the new strands. A topoisomerase helps relieve the tension in the double helix caused by the unwinding. The two DNA strands in the double helix are replicated at the same time, giving rise to two identical DNA double helices, each consisting of one old and one new DNA chain. Both new strands are synthesized in the 5’ – 3’ direction, but the two DNA polymerase III holoenzymes move in the same direction, which means that one of the template strands has to loop back through the replication complex. This strand is synthesized discontinuously (in pieces or Okazaki fragments) and is called the lagging strand. The other strand, which is synthesized continuously, is called the leading strand.
DNA polymerase III cannot start synthesizing a new piece of DNA, it can only continue synthesis. This problem is solved by primase, which synthesizes a short RNA primer that can be used as a starting point for DNA polymerase III. The primase is together with the helicase and topoisomerase called the primosome.
Another DNA polymerase, DNA polymerase I, chews up the RNA primers and synthesizes DNA instead. Finally, the gap between the DNA sequences is sealed by DNA ligase.
2. PCR (Polymerase Chain Reaction) is a very commonly used molecular method.
a) Explain (with figure) how PCR works.
b) Think of a specific situation where PCR could be used.
a) PCR is a method to amplify (make many copies of) a piece of DNA. To do this you need:
I. DNA template (could be for example genomic DNA from a bacterium)
II. DNA primers specific for the piece of DNA you want to amplify
III. DNA polymerase (must be heat-stable to withstand the high temperatures)
IV. Deoxynucleotides (dNTPs)
V. “Temperature cycler”, but you can also use three waterbaths/heatblocks set at the different temperatures and move your tubes around manually!
The figure below shows a typical PCR reaction, where the DNA strands are first melted apart at 95°C, primers annealed (base-paired) to the template at 50°C and the new strands synthesized by the heat-stable DNA polymerase Taq polymerase at 72°C. The temperature is then raised to 95°C again, and the three steps are repeated a number of times, typically 20 cycles.
Since the newly synthesized strands from each reaction cycle are also recognized by the primers, they will be used as templates in the next cycle. For every cycle you run you will double the number of copies of your DNA piece, which means that if you start with just one DNA molecule and run a PCR with 20 cycles you should end up with 220 (over one million) copies!
PCR reaction
b) PCR can be used for many different purposes, ranging from diagnosis of infectious diseases to solving crimes. The greatest advantage with the method is (as mentioned above) that only tiny amounts of template (DNA starting material) are needed. Some of the specific applications of PCR include:
- Evolutionary studies: PCR and sequencing of DNA isolated from remains of extinct organisms can be used to investigate relationships between different species.
- Crime solving: Parts of the genome known to vary a lot between people can be PCR amplified, analyzed and tie a suspect to a crime scene.
- Diagnosis: Infectious diseases can be detected in a blood sample by amplifying a gene characteristic for the pathogen suspected to cause the disease, for example a virus or a bacterium, without having to culture the pathogen first.
3. Plasmids and restriction enzymes are, as most available molecular tools, naturally occurring in bacteria.
a) What are they used for in bacteria?
b) In what way can you use them in the lab?
a) Restriction endonucleases are a large class of enzymes that recognize and cleave certain DNA sequences specifically. The recognition sequence varies between different restriction enzymes from different bacteria. They are used by bacteria as a kind of immune system for protection against phages (viruses infecting bacteria) infections, where the viral DNA is cut at the sequences recognized by the endonuclease and then destroyed by the cell. To avoid destroying its own DNA, bacteria have enzymes that methylate the sequence recognized by their specific set of restriction endonucleases. The endonucleases cannot cut methylated DNA, and thereby will only cut viral, unmethylated DNA.
b) Restriction enzymes are very useful in the lab. They recognize and cut specific DNA sequences, which means that you can for example cut out a gene from one plasmid and “paste” (using a ligase) it into another plasmid. You can also make use of their sequence specificity to identify a variation (mutation) in the recognition sequence, where you will not get a cut if a nucleotide has been changed. If you treat a piece of DNA containing the recognition sequence with the corresponding restriction endonuclease and separate the cleavage products by size on an agarose gel you can see if you get two short fragments (cut – no mutation) or one long fragment (no cut – mutation in the recognition sequence).