Primers, DNA and enzymes – it’s a PCR! by Leighvi Pilcher
A PCR – Polymerase Chain Reaction – is a reaction that is used to amplify the genes from a sample of DNA. In my PCR, we were trying to identify the normal “wild type” samples, and the SOD samples. I ran 12 samples in total – 2 controls (1 wild type, 1 SOD) and 10 random samples.
The first step in a PCR (aside from acquiring the DNA – this is done by taking a sample, and digesting it in Proteinase K, which is an enzyme, and heating it at a high temperature) is making the reaction mixture. Within the reaction mixture, 4 primers are used. 2 of these primers are complementary to the wild type DNA, and the other 2 are complementary to the SOD DNA. dNTPs (free nucleotides) are also added. These bond to the newly amplified single DNA strands in order to make them double stranded again. A buffer is added to maintain the pH of the reaction mixture, with magnesium chloride added in order to remove the additional phosphate molecules from the dNTPs. Taq polymerase is added, which copies the target DNA. All of the reagents of the PCR mixture are added to sterile water, and enough is added to make 14 samples. After each addition, the tube is vortexted, which ensures that the mixture has been evenly mixed.
Once the reaction mixture is made, it is separated into 12 Eppendorf tubes. After this, the samples are added. Again, these are vortexted, which ensures an even distribution. The samples are labeled, and added to the PCR machine. In the PCR machine, the samples are heated. The heat “melts” the hydrogen bonds in the DNA. They are also cooled in the machine, which subsequently allows the hydrogen bonds to reform. After this, the samples are heated to 75 Celsius, which is the optimum temperature for taq polymerase, so the target DNA can start to be copied.
After running the PCR machine, a process called electrophoresis is carried out. Agarose powder is added to a buffer solution so that 10ml of a solution is formed. 50ml is used in the gel, and it is heated to approximately 100 Celsius before cooling to 60 Celsius. This is added to the electrophoresis chamber, which has slides put in it to help identify the holes that the PCR samples will be added to. Once the gel has set, the combs are removed, and the remaining buffer is placed on top. Bromophenol blue is added to each of the 12 samples, as this helps to identify them when placed under UV light in the transillumiantor. A control ladder is added to the first chamber in the gel, which is used to show the approximate placement of the DNA bands. The remaining chambers are loaded with the 12 samples. Once this is done, the gel is “run”. In this process, electricity is passed through the gel (50 volts) for 30 minutes. This stimulates the movement of the samples. The denser bands rest closest to the chamber, with the lighter bands resting further away from the chambers.
Now that the hard part is out of the way, the samples are viewed in a transillumiantor. Here, a UV light is beamed from under the samples, which shows the movement of them. As you can see, the far left of the image is the ladder. Next to it is the wild type control, with the SOD control next it it. Samples 1, 2, 4, 6, 7 and 9 were all wildtypes, and the remaining samples were SOD samples.
When I was first given all of this information, I screamed internally, because it is such a lot to take in. It’s not necessarily a complicated procedure, however it is long and fiddly. So, I summarised in the following steps:
- Reaction mixture
- Addition of samples
- PCR machine
- Gel preparation
If only it was that simple!
Leighvi undertook her placement in UCL’s Sobell Department of Motor Neuroscience and Movement Disorders, at the Institute of Neurology, supervised by Dr Bernadett Kalmar in the group of Professor Linda Greensmith.