Inside the test tube

Making use of his vast knowledge on DNA and of all the laboratory equipment available in the Eighties, Dr. Mullis started carrying out experiments to carry through the brainwave he had on the path that evening. His aim was to define a method that allowed him to copy a specific piece of DNA many times ‘in vitro’, i.e. in a test tube. The PCR method works on a theoretical principle that we will now describe.
With a bit of imagination we can compare the implementation of a PCR laboratory experiment with the preparation of a cake. To start preparing the cake we arrange the required ingredients on the table (eggs, milk, butter, baking powder, flour, etc.) and then we mix them in a bowl. In the case of the preparation of a PCR reaction we arrange the various biological and chemical components (the cake ingredients) on the laboratory counter in their proper containers such as test tubes, pipettes and jars. Some preliminary steps must be carried out before starting the preparation of the cake such as dividing the egg yolk from the albumen, or working at the butter when it is too cold. In the case of a PCR too some preliminary steps must be carried out before mixing everything in just one last test tube (the mixing bowl).
DNA extraction
First of all we have to extract the DNA from the nucleus of the cell in which it is contained. For example the DNA must be extracted from bacterial or blood cells or from the remains of a human or animal mummy. The procedure entails various stages with some chemical compounds and subjecting the cell sample to suitable treatments. In other words we have to demolish the cell structure, i.e. break all the cells of the sample so as to make the DNA come out. Then it is necessary to digest the proteins that are associated to the DNA molecule, this means that at the end of the procedure a solution that contains only the DNA that has been separated from the digested proteins is obtained. These proteins have been transformed into amino acids (the bricks that make up proteins). At the end of this initial phase we will have a test tube with DNA dissolved in a liquid such as ethanol. In this environment DNA is not soluble and its filaments are visible in suspension in the test tube.
The components of the PCR reaction
These are the ingredients of the cake when they have not been mixed yet. On the laboratory table we have the extracted DNA with the exact sequence that has to be copied which is called the target and the aim of the experiment is to multiply this specific piece. From now on we will use the ‘convention’ that the sequence that has to be copied is called ‘target’ so that it will not be confused with other DNA fragments that take part in the PCR.
In addition two more fragments of DNA called primers are used; their function is to provide a starting place for copying and replication of the target sample, i.e. they help to trigger off the beginning of the replication. During the PCR reaction, the target filament is heated and its double helix unwinds: two single strands of the DNA target are obtained in this way. Here the primers are added and they bind to the single stranded DNA, in practise they arrange themselves close to each single strand. This is the starting place for replication.
But for replication of the target to begin, we need yet another ingredient, DNA polymerase that, as we have already seen, is a cell enzyme. DNA polymerase also has the function of replicating and repairing DNA. This enzyme can elongate a primer by adding nucleotides (that are the bricks that make up a DNA strand) one at a time. In the recipe, a mixture of small, loose bricks (the basic structures of the DNA filament) must still be added; these nucleotides will be used to build up the new DNA filaments. We have already described what nucleotides are in point 1.
A mixture of other supporting elements (such as salt and grated lemon peel in a cake) must also be added, such as magnesium that helps to create the right chemical conditions for the reaction to take place.
The thermocycler
This is the oven to cook the cake in. We have all the necessary ingredients and so we mix them together in one test tube just like we pour the cake mix into a cake tin.  To bake the cake we put the tin into the oven and cook it for a fixed time and at a specific temperature, for the PCR we put the test tube containing all the compounds into the thermocycler. This laboratory equipment has a thermostat: thus it is possible to regulate and programme the temperature variations and length of time of each of the three phases of the amplification, i.e. the three steps that are necessary to copy the target sample many times (the baking of the cake). The functioning of the thermocycler is based on different systems of heating and cooling by means of air, liquids, electrical resistances etc.  The thermocycler has a small drawer in which the test tubes where the PCR reaction is going to take place are inserted; there are different types on sale, of varying sizes depending on the requirements of the laboratory.

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