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Looking for DNA
The discovery of the PCR method
Everyone knows the novel or has seen the film Jurassic Park in which, thanks to laboratory techniques, the blood of a dinosaur preserved in the buccal organs of a mosquito trapped in amber is extracted and so it becomes possible to reproduce the DNA of the long extinct animal. The scientists that work in Jurassic Park used the PCR method to bring to life dinosaurs that had been extinct for millenniums, from eggs produced in the present thanks to the DNA found in the mosquito! How was this possible?
Around the beginning of the Eighties in the world of biological sciences there is another powerful revolution. The setting is always a biochemistry, molecular biology or microbiology laboratory and the main character on which we are focusing our attention is still the DNA molecule. Few inventions have revolutionised the course of molecular biology as much and as fast as PCR, the acronym for Polymerase Chain Reaction. The polymerase chain reaction of DNA is a molecular biology technique that enables the multiplication, i.e. the copying (technically the ‘amplifying’), of a specific chain of DNA. When we talk about a specific chain of DNA we mean a fragment of nucleic acid, of which the initial and final nucleotide (the series of building blocks that make up the DNA filament) sequences are known, in other words the beginning and end of the fragment. This replication process takes place normally in nature, in the cells of our body, when the DNA contained in the nucleus unwinds from its double helix conformation and its filaments are copied by the DNA polymerase enzyme. This ordinary phase of cellular reproduction is imitated in a test tube by the PCR technique. In practise, a piece of ‘complete’ DNA with a double helix is reconstructed from a strand with a single helix. The revolutionary fact is that using this laboratory technique, man can choose to take a particular part of DNA that interests him and study and utilize it in compliance with the purpose of his research, outside the cellular mechanisms that take place in a living body.
The utilization of PCR therefore enables the ‘amplification’ of a specific tract of DNA, multiplying i.e. reproducing it, billions of times in a very short time, a little over an hour, and obtaining a great number of copies.
This laboratory technique, worked out in the first half of the Eighties by the brilliant mind of Dr. Kary B. Mullis, has enabled us to better understand the genetic information contained in the DNA of each individual, with crucial consequences in wide-ranging fields, from laboratories where pure research is carried out to hospital analysis laboratories and to courtrooms. A confirmation of the importance of this achievement is the Nobel Prize for Chemistry that Kary B. Mullis received in 1993.
Before trying to understand how a PCR ‘reaction’ (this is how a procedure that takes place in a test tube is called) applied to a specific tract of DNA works, let us learn about the life and the personality of its inventor, a charming genius of our times.
Since when he was at the Dreher High School of Columbia (USA), Dr. Mullis proved to be an impertinent youngster gifted with a strong sense of humour and soon became a leader among the students. In the years that followed he accumulated academic titles and important assignments and devoted himself to paediatrics, biochemistry and cardiology. In 1979 he worked for the Cetus Corporation of Emeryville, California and as a chemist specialized in DNA he worked at a research on the synthesis of oligonucleotides, i.e. the building blocks that compose DNA and he invented the polymerase chain reaction (PCR).
He had the real intuition about how to multiply DNA billions of times in 1983. The legend narrates that Dr. Mullis was going home from the laboratory on an evening like many others and his mind was lost in hazy thoughts but suddenly he noticed that the path he was walking on was made up of many sections arranged one after the other like the rungs of a ladder. He went on walking and the path divided at a junction. This reminded him of the two strands of DNA that divide before replicating, and then, suddenly, the street lights that lined the path came on one after the other because it was getting dark. The brainwave hit him in a second: a way to make infinite copies of DNA strands from a single portion came to his mind. He turned around and ran back along the illuminated path to the laboratory to try and put his ingenious idea into practise.
Dr. Mullis’ career continued brilliantly, he received the Nobel Prize and another very prestigious prize, the Japan Prize. He worked on both technological researches and on those regarding the photochemistry of DNA. In one of his most successful books, ‘Dancing Naked in the Mind Field’, written in the year 2000, his sense of humour and brilliance can be appreciated in disparate sections: he writes about poisonous spiders, about science and parapsychology, about astrology and the Hiv virus.
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.
Finally it is time to bake the cake; we take the test tube and insert it in the thermocycler. What happens next? The baking consists of subjecting the test tube to a certain number of thermal variation cycles. Each thermal cycle is made up of three phases. Periods of heating are alternated with periods of cooling.
1st phase: denaturation
In this first phase of a PCR reaction the total separation of the two DNA filaments of the target sample that has to be replicated takes place. Usually the temperature applied to the test tube must reach 94°C for 30-60 seconds. By warming the DNA the unwinding of the double helix and the separation of the two filaments takes place; it is said that the DNA is denatured.
2nd phase: hybridization of primers
This is the most delicate moment in which the primers have to ‘stably hybridize’ to the DNA template. What does this mean? It means that the primers that have been added to the mixture have to bind to the single separated strands of the original DNA target sequence. Usually the procedure involves various experimental attempts in order to find the ideal temperature that promotes the binding of the primer strands to the target strands according to the type of DNA sample that has to be replicated. Usually the temperature is lowered to about 30 – 55 °C.
3rd phase: the extension of the new DNA strand
The ideal temperature for polymerization, i.e. the construction of DNA filaments identical to the DNA target depends on the DNA polymerase enzyme utilized. At present an enzyme called Taq polymerase is used and a temperature of 65 - 72°C is applied for 5 minutes. In this phase the polymerase enzyme physically attaches the building blocks of the filament (the nucleotides) to the primers that are coupled and complementary to the target filament. In practise, the copying of each single target filament takes place. DNA polymerase takes care of correcting any mistakes in the event that a ‘wrong’ nucleotide is incorporated, an eventuality that can take place since the mechanism is never perfect. Thus the DNA polymerase is capable of eliminating an error and creating a correct pairing with the corresponding nucleotide present on the filament that is being copied and replicated.
During the first cycle of the PCR reaction new filaments are synthesized which, after denaturation i.e. the division of the double helix and separation of the parallel strands, can bind themselves to the primers. These products accumulate arithmetically but it is only from the second cycle on that two single helix products are formed that will make up a fragment identical to the double helix target DNA.
Thus with repeated denaturation cycles, hybridization of primers and their extension an exponential increase of the copies of the target DNA segment is obtained.
During each ‘cooking’ cycle the number of fragments of DNA that have to be copied (i.e. those present between two primers) doubles. After just 32 repeated cycles, millions of copies of double helix DNA fragments identical to the target are already formed.
At this point the cake is ready and we can take it out of the oven. It will have risen and cooked and will be ready to be tasted. In the same way the test tube removed from the thermocycler at the end of all the thermal cycles of ‘cooking’ and cooling to which it has been subjected, is ready, the PCR is over. The numerous copies of DNA, identical to those of the original sequence we wanted to replicate, are available to be subsequently analysed and utilized in different ways according to the requirements of the experiment.
written by Eliana Marchisio
