PCR – Polymerase Chain Reaction is one of the routine laboratory technique. It is used to make copies of desired DNA, just like photocopier machine that makes copies of desired document. Desired DNA could be a gene or segment of DNA on which the experimenter or scientist is working. The motive of using PCR is to produce clones or copies of DNA which can be further used sequencing or cloning. PCR is used in Forensic Science, Diagnostic labs, Molecular Biology and different branches of life sciences. This technique was developed Karry Mulis in 1980s and he received Noble prize for his contribution in 1993.
The PCR technique can be called as in-vitro DNA replication. Hence, the PCR techniques needs all DNA replication’s enzymes and proteins. In-vivo, the cell replicates its DNA and produces its daughter DNA before division. Hence, in-vivo, cell produces only one copy of its DNA. While in PCR, we can produce many copies of the DNA replication.
Requirements or ingredients for PCR –
- Template DNA – it is the desired segment of DNA which needs to be amplified. The DNA can be of any source like plant, animal or microbes. The DNA could be genomic or plasmid. To amplify the DNA, 1 to 50 ng minimum concentration of DNA is required. The desired segment of DNA could be the sample for paternity test, forensic studies, cancer studies, detection of pathogenic microorganism etc.
- Taq DNA Polymerase – DNA polymerase enzyme synthesizes new daughter DNA strands by reading the parental or template strand. In PCR, special type of enzyme is used called as Taq polymerase. It is isolated from Thermus aquaticus, it is an extremophile which lives in hot springs and can grow upto 80°C. Due to its high optimum temperature, its DNA polymerase is heat stable and active around 70°C. In order to make copies of DNA, the DNA need to be unwind, melt or separated. In vivo, it is done by helicase but in PCR, its unwinding is carried out with the help of high temperature (70°C). At such a high temperature, human or E.coli DNA polymerase is ineffective or non functional. Therefore, Taq DNA polymerase serves to be best polymerase enzyme for PCR technique.
- PCR Primers – Like human or E.coli DNA polymerase, the Taq DNA polymerase also needs primer to initiate DNA synthesis. Primer is a short nucleotide sequence that binds at 3′ end of the template. DNA polymerase reads the template strand and extends the primer by adding nucleotides at 3′ end of the primer. In PCR reaction, the scientist can choose the region of DNA that needs to be copied or amplified and based on that the primers are artificially designed. As we need to amplify both the strands we need two primers. They are complementary to the flanking region of the desired gene present on template.
- Free Nucleotides – Deoxynucleosidestriphosphates are added in the mixture like dATP, dCTP, dGTP and dTTP because they are building blocks of DNA. They are added in equimolar concentration.
- Magnesium ions – The magnesium ions are added in the form of Mgcl2 solution. The magnesium ions are required as cofactor for the DNA polymerase enzyme.
- Buffer – The PCR reaction is carried out in suitable buffer that promotes DNA synthesis. The buffer maintains the pH from the range of 8 to 9.5. It contains potassium and ammonium ions. The potassium ions promotes the binding of primer to DNA template and ammonium ions avoids mismatched primer – template binding hence increases the efficiency.
Steps of PCR –
The mentioned ingredients are added in the PCR tubes and the tubes are loaded in the PCR.
- Denaturation – it is the first step of PCR. The DNA helix is formed due to the hydrogen bonds between complementary base pairs. The denaturation means unwinding of helix and separation of DNA strands. In PCR, the DNA strands are separated by the heating the PCR mixture at 96°C. The denaturation provides single stranded DNA which is required for synthesis of new strand.
- Annealing – It is the second step. Annealing means binding of primer to DNA template. In order to bind the primer to template, the temperature of the reactions needs to be cooled to 55 to 65°C.
- Extension – The Taq polymerase enzyme extends the 3’OH end of the primer by adding the free nucleotides and synthesizes new strand. The reaction occurs at 72°C.
These three steps makes one cycle. Such cycle is repeated 25 to 30 times (or based on requirement). The duration for PCR depends upon the number of times the cycle is repeated. The new synthesized DNA also serves as template for second cycle. Hence, the DNA growth or multiplication pattern is of exponential type. The operator can feed and set the required number of cycles in order to obtain required copies of DNA. The PCR results in the synthesis of millions of copies of desired DNA.
Visualization of amplified DNA –
The amplified DNA can be observed in gel electrophoresis. It is technique in which DNA runs in the gel under the electric force. The running speed of the DNA depends up on its size. In order to visualize DNA, it is stained with Ethidium bromide dye. It appears in the form of band. The size of the DNA can be guessed by comparing it with ladder. The ladder consist of DNA fragments of different and known size.
Application of PCR –
The PCR is used in different branches of Life Sciences –
- It is used for DNA sequencing. Because for sequencing, we need many copies of desired DNA.
- It is used for cloning. Where we amplify the desired gene and insert it in vector. And the vector is then transformed in the target cell.
- It is also used in Medical Diagnostics. Where the patient’s sample is amplified and tested.
- It is used in Forensics, where the DNA is isolated from the blood sample obtained from crime scene and amplified.
You can watch the animation for better understanding –