Hope you have read our previous article “What is PCR?” which gives a better foundation for this one too. Today let’s find about the technical background in the Polymerase Chain Reaction so-called PCR.
As mentioned in the previous article PCR Is a technique that use to make multiple copies of a specific DNA region. This technique can be used to directly amplify a specific sequence of DNA molecules (or specific DNA sequences in a complex mixture) into millions of copies in a short time.
To do this, these factors are needed.
- Sample/Template DNA
- DNA polymerase enzyme – Taq polymerase (let’s see why later)
- PCR primers
- dNTPs ( Deoxynucleoside triphosphates) – A, G, T, C
- Buffer solution
First, we need to extract out the sample DNA or DNA region that needs to be amplified by polymerase chain reaction( PCR ). DNA extraction can be done using various methods. That will be discussed later.
DNA polymerase enzyme is required for this process. Here we use Taq polymerase as the polymerase enzyme. There is a reason behind the use of Taq polymerase. Enzymes are made out of proteins. Which are highly sensitive to temperature. They get easily denatured at high temperatures and they will not come back to the previous state again. In the process of PCR, we have to use high temperatures as 95-96 C degrees to denature the double-stranded DNA sample. Therefore the polymerase enzyme that we use needs to be heat stable. Taq polymerase has been isolated from a bacterium that lives in hot springs, Thermus aquaticus . Therefore the enzyme is not will be denatured by the high temperature.
Primers are short synthetic single-stranded DNA molecules. Those will provide the starting point for the polymerase to work and synthesis the new strand. Two primers are used in the PCR reaction. Those are forward primer and backward primer. These primers are made by looking at the complementary bases of the targeted DNA region.
All these 5 ingredients are then placed in the PCR machine. That is actually a thermal cycler that changes the internal temperature of the machine at a programmed time frame. At different temperatures, different steps of the PCR are happening. Let’s see what are those.
There are three basic steps in the PCR reaction.
First, the temperature rises to 95-96 0C. At that temperature, double-stranded DNA is separated and gives two single-stranded DNA. Then the temperature lowers to 55-65 0C. At this temperature, the primers anneal to the template DNA strands. This is the annealing step. Then again temperature rises to 72 0C. This is the optimum temperature for taw polymerase action. Therefore it begins to synthesize new strands. After about 1 minute. The temperature is again raised to 95-96 0C and cycles the process. This process can be run until we get enough copies. At each round, the number of copies of the sequence between the primer sites is doubled. Results of a PCR reaction are usually visualized using gel electrophoresis.