Polymerase Chain Reaction (PCR)
It is one of the most important biotechnological tools developed. It refers to a biological technique that helps to produce several copies of DNA outside of any living cell. DNA polymerase is the key enzyme that is present behind the whole process. The enzyme involved in the synthesis of new DNA strands by binding with a single DNA strand. This technique was developed by Kary Mullis who was awarded the Nobel Prize in 1993 for this achievement. The development of recombinant DNA technology is mostly dependent on this technique.
Principle and Working Mechanism
The main objective of using a PCR is to produce a huge number of DNA copies. Therefore, template DNA molecules are the first essential component of the whole process. Apart from that primers are also an important component that binds with the template DNA. The reaction mixture also contains all four deoxyribonucleotide triphosphates i.e., dATP, dCTP, dGTP,dTTP and DNA polymerase.
The major steps of PCR can be divided into three parts:
Denaturation at 940C
This is the first step of the process when the temperature is maintained at 940C. At this temperature, the DNA double helix is converted to a single strand and other enzymatic reactions such as the extension of DNA from a previous cycle is arrested.
Annealing at 540C
Then the temperature is reduced to 540C and the primers present at the reaction mixture started to get attached with the template DNA molecule. Due to the low temperature, the bonding between the primer and template occurs. The primer helps the polymerase to find out its attachment site.
Extension at 720C
It is the optimum temperature for the polymerase. At this temperature, the polymerase starts working. The synthesis starts from 5’ end and moves towards 3’ end. The polymerase helps to join the nucleotides at the complimentary position to the template DNA. As a result, another copy of DNA is produced. The whole process goes for 30-40 cycles which leads to amplification of the template DNA into billion copies. These copies are then further analyzed.
Constraints in PCR
The type of polymerase generally used in PCR is Taq polymerase. This enzyme is isolated from Thermus aquaticus which is a thermophile bacteria and due to the nature of the bacteria, the enzyme can withstand more heat than other types of a polymerase. A major disadvantage of this type of polymerase is that it lacks 3’ to 5’ exonuclease activity. Therefore, it is impossible to find out whether the nucleotides are correctly inserted or not. As a result, the error rate can be distinctly high. However, this problem can be countered by using other types of polymerase enzymes, isolated from other organisms such as Thermococcus litoralis which has exonuclease activity.
The DNA amplified in a PCR is generally of a size of 2-3 kb, it is not possible to complete the amplification process in case of larger DNA. The polymerase fails to complete the DNA extension in larger DNA molecules. Apart from that, the typical heating cycle is not optimum to complete the polymerization. This problem can be solved by using a slow heating cycle and different polymerase.
It is one of the major reason behind the errors occurred during the amplification of DNA in PCR. The primer often attaches to different sequences due to sequence duplication and there is no system available to check whether the primer is attached with the specific sequence. As a result, the gene of interest often left alone and the other parts are amplified. To reduce this problem, many software are developed to design a particular primer specific for the gene of interest.
Practical Modifications of PCR
Nested PCR is developed to reduce the non-specific binding of the primers. In this case, two sets of primers are used in two cycles of PCR. The first set of primers amplified the template DNA present in the reaction mixture while the second primer is specific for a secondary target which is present at the first amplified part of the DNA. As a result, the gene of interest can be amplified properly.
This type of PCR is used when only one known internal sequence is present. One important application of inverse PCR is to find out various insert locations. For example, several retroviruses and transposons randomly attached to the genomic DNA. Therefore, the determination of the specific insert can be performed by using primers designed from the internal known sequence. Inverse PCR is characterized by a series of digestion and self-ligation which in turn helps to find out the known sequence at either end of the unknown sequence.
RT PCR stands for reverse transcription-polymerase chain reaction which is a modified type of PCR used to convert known sequence of RNA to DNA by reverse transcription and the DNA sequence is then amplified for further analysis. The aim of using this type of PCR is to measure the amount of a particular RNA. It can be measured by monitoring the amplification by quantitative PCR or qPCR. RT-PCR and qPCR are important tools to study gene expression and quantification of viral RNA ina laboratory setting.
This type of PCR is used to amplify one strand of the DNA than the other. In many cases, only one strand of the DNA needs to be amplified and asymmetric PCR helps to obtain the result. The PCR reaction takes place normally but the primers used for amplification is different from the general type of PCR. A major disadvantage of this type of PCR is its slow amplification rate as a result of which several cycles are required to complete the PCR process.
Touch Down PCR
This type of PCR modification helps to avoid non-specific attachment of the primers. The primer requires a specific annealing temperature to attach to the specific sequence. In this case, the temperature is increased rapidly and then reduced step by step to obtain specific primer attachment.
Applications of PCR
Human Health and the Human Genome Project
PCR is an essential tool that can be used to improve human health and life. In medical science, PCR is used for the detection of infectious organisms and the detection of mutation in various genes. In the case of the detection of diseases like AIDS, PCR can be used to directly study the virus DNA and it is more specific than the standardized detection done by ELISA. Moreover, PCR has high potential in the application of detection of diseases like Lyme disease, where it can directly identify the presence of bacterial DNA in joint treatment. PCR is also used for the detection of Helicobacterium pylori and sexually transmitted virus diseases. The human genome project refers to the study of all human genes. PCR plays an important role in this project as it helps to identify specific genes along with mutations and rates of mutations in those genes.
This is technique is used in forensic science to compare the DNA of a person with a given sample such as a blood sample found in a crime scene. The sample may contain a very small amount of DNA. The application of PCR comes at this part to amplify the small amount of DNA from samples like blood, semen, saliva, hair, etc. The amplified DNA fragments then further analyzed by gel electrophoresis.
Detection of hereditary disease
It is difficult to understand hereditary diseases due to its direct connection with the genome. This complex and difficult process can be easily analyzed using PCR. The PCR can amplify the DNA and by using a specific marker, the particular gene can be detected which is responsible for the disease. Apart from that, the mutation rate in that specific gene can be also be analyzed by using PCR.
Gene cloning has different applications in industrial as well as laboratory scale and PCR can be used for specific gene cloning. When a particular gene of interest needs to be cloned, PCR is used to amplify the gene. It is then inserted in a vector and the vector is then further transported the gene inside a cell. Therefore, PCR is required to complete the gene cloning studies.
Analysis of ancient DNA
PCR can be used in the analysis of ancient DNA. For example, the DNA isolated from Egyptian mummies can be amplified in PCR for further studies and identification of the person. The gene can be used to compare with the gene of recent time and the evolutional analysis can be also be performed.
- Bioinstrumentation by L. Veerakumari, MJP Publishers.
- Garibyan, L. and Avashia, N., 2013. Research techniques made simple: polymerase chain reaction (PCR). The Journal of investigative dermatology, 133(3), p.e6.