Polymerase chain reaction (PCR)
Polymerase chain reaction, in simple words, polymerase chain reaction, or PCR, is a technique that is used to make several copies of a small fragment of DNA or RNA. PCR is made up of two words: polymerase and chain reaction. Polymerase means an enzyme that makes polymers of any other molecule. In this technique, the DNA chain reaction is a type of chemical reaction that progresses in an exponential way, or in simple terms, if the first reaction produces two molecules, the second reaction will make four, the third will make sixteen copies, then 32, 64, 128 512 copies, and so on. So, in a matter of just a few hundred reactions, we can produce billions of copies of a single fragment of DNA before progressing further. Why would we want to make a billion copies of a region of DNA? PCR has thousands of users in diagnosing infections and infectious diseases. For example, it is used to diagnose infections by viruses. We take the blood of a patient that has been infected by the virus and then we amplify the DNA of the virus so that we are able to study which type of virus it is and its properties. It, like PCR, has been used in hundreds of other fields such as crime investigation, genetic research, molecular biology, and so on. The most commonly used equipment in PCR is the thermal cycler, also known as the PCR machine. Inside the PCR machine, we have these small tubes in which all the chemicals are inserted and the reaction takes place. The key ingredients of a PCR reaction are tagged polymerase primers, DNA templates, and nucleotides. TAC polymerase is a type of DNA polymerase, and DNA replication in any organism is the same.PCR requires a DNA polymerase enzyme that makes new strands of DNA using existing strands as templates. The DNA polymerase typically used in PCR is called dark polymerase. The heat-tolerant bacteria from which it was isolated was named thermos aquatics. This material lives in hot springs and its DNA polymerase is very heat stable and it is most active around 70 degrees. This heat stability is ideal as high temperatures are needed for this reaction. The second important thing that we need to perform PCR is the primers. DNA polymerase needs primers to start the reaction, as the polymerase cannot initiate this reaction but can only propagate it. PCR primers are short sequences of nucleotides, usually around 20 nucleotides in length. Primers provide a starting point for DNA synthesis. Primers are also important as they help to select the exact portion of DNA that will be amplified. We use such primers in each PCR reaction and they are designed so that they answer late to the target region of the DNA to be copied. The DNA template is the segment of original DNA that we want to amplify and nucleotides as we already know are the basic building blocks used for DNA synthesis.
PCR procedure
PCR involves three simple steps.
- Denaturation
- Annealing
- Extension
In the first step of PCR, we raise the temperature of the machine to 96 degrees Celsius. This is done to denature or separate the two strands of DNA, so as a result of this step, we get two separate strands of DNA. The next step of PCR is known as annealing. The meaning of the word annealing was used in the metal industry where they heat a certain metal to a higher temperature and then cool it. This process is called annealing, which is basically done to remove the internal defects from the metal. The same is the case here in PCR where we first raise the temperature of the machine to 72 degrees for this step one, which is denaturation, and then we cool the temperature to 55 degrees so that the primers in the PCR tube can bind to their target sequence on the single-stranded DNA. For example, if a patient suspects an HIV infection, we take the blood sample of the patient and perform PCR on it. The blood sample contains the virus and the virus's genetic material, the DNA, so our aim here is to confirm whether the patient has the infection or not, but the DNA of the virus in our sample is a very small quantity. So we need to amplify the DNA of the virus to detect it. Perform the first step of PCR, which is denaturation, to separate the two strands of the DNA. This is the denatured DNA of the virus in our sample, and in green, we have a specific sequence or gene that we have decided to amplify and detect later. Use two primers, which are basically used to mark the specific area of the DNA that we want to amplify. The primers have a sequence that matches the sequence that is present at the starting point of the target region of these two strands of DNA. The primers bind to the template DNA by complementary base pairing. In this way, we are able to select a particular region of the DNA that we want to amplify. This was annealing. Next comes the third step, known as extension. It is basically used to make the new DNA again. Extension: We increase the temperature again to 72 degrees so that the tag polymerase extends the primers, synthesizing new strands of DNA. The tag polymerase adds new nucleotides to the short sequence of the primer to form new strands of DNA that are complementary to the original strand. These ingredients are assembled in a tube along with cofactors needed by the enzyme and put through repeated cycles of heating and cooling that allow new DNA to be synthesized and get two new double-stranded DNAs. One is the original parent DNA and the other one is a new DNA formed by the TAC polymerase. This process is repeated again and this makes the PCR a chain reaction basically. The products of the first reaction are used as the substrates for the next reaction. When we cool down the temperature to 55 degrees for annealing, we will get four single-stranded DNA. The TAC polymerase comes in for extension and this time it leads to the synthesis of four new single-stranded DNA because the substrates for the enzyme are also doubled from the second cycle of the PCR. We get one, two, three, four, five, six, seven, and eight new strands of DNA. These steps are repeated again and again and this is what makes the PCR a chain reaction to summarise basically. The products of the first reaction are used as the substrate for the next reaction, which means that if in the first reaction we got DNA out of one, in the second reaction we will get four DNAs out of two, and in the third reaction. Due to the exponential nature of the change reaction, we are able to produce billions of copies of the same DNA fragment from only a single copy of the fragment. This is repeated 25 to 30 times in a typical PCR reaction, not a hundred times, which takes two to four RNA fragments depending upon the length of the DNA region being copied. Due to the exponential nature of the change reaction, we are able to produce billions of copies of the DNA fragment from only a single copy of the fragment. After this, the temperature is again lowered to 15 degrees so that the products of the reaction can be stored to check whether the PCR has generated the correct products. We use a technique known as agarose gel electrophoresis. The process of PCR takes place at the end.
PCR is an amazing technique that has revolutionized the field of diagnosis and research.
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