Introduction:
PCR Polymerase chain reaction is a method which came about in the 1980s and is widely used In scientific research today in the world. The polymerase chain reactions allows us to amplify a certain gene, a certain sequence of a DNA fragment very quickly so that we can produce millions of copies for that specific type of DNA. There are 4 main introductory steps that are needed to carry out a polymerase chain reaction e.g. The target DNA strand which is needed to replicate, a pair of DNA primers are needed which range of nucleotides that aid in the replication process, we also need a heat resistant DNA polymerase because of the protein complex which moves along the DNA and helps in replication and lastly we need the deoxyribonucleotide triphosphates. The mechanism of PCR and how It actually works consist of 3 steps – denaturation, annealing and elongation.
Mechanism of PCR:
The process begins with denaturation, where the target DNA fragment is taken and the sample is heated to 96 degrees Celsius to break the hydrogen bonds, this process takes place for a few seconds. After the bonds are broken the DNA strands then separate, after this the temperature is lowered so that hybridization can take place to help the primers which are added in the solution to attach to the specific DNA strands. The structure of the primers are complementary to the sequence of the DNA which they attach to, this is the second step known as annealing. One DNA primer binds to the 3’ end of one strand and another binds to the 3’ end of the complementary strand. Then the last step takes where the temperature is once again raised to about 72-74 degrees Celsius, the reason the temperature is raised again to 72 degrees Celsius is because that is the temperature of the heat resistant DNA polymerase, this is when the DNA polymerase will bind onto the DNA and will begin adding the deoxyribonucleotides triphosphates molecules and begin to synthesize and elongate the DNA. At the end of step number three which is elongation, we have formed two identical copies of the DNA that we began with. After the cycles repeat again and again the number of copies of the DNA sample keep increasing in number. After an hour we can make millions and millions of copies of the DNA sample. This is why the PCR is widely used because of its efficiency and effectiveness.
After the PCR is done to actually visualize those results we need to perform a gel electrophoresis. This is the technique where all the DNA fragments are separated according to their size through a gel matrix which pulls them through an electric current. Usually a control so first set up which we can compare our results with later, this is called a hyperladder. The DNA fragments then form bands on the gel, this can be viewed on the gel with the use of a stain/dye which binds to the DNA. The DNA fragments are put on the gel in order next to the hyperladder, so that the bands can be distinguished by how many base pairs they produced (INABA, 1989).
Types Of PCR:
There are specific types of the PCR assay:
Reverse transcribed PCR (RT-PCR):
In many applications of PCR, the starting template material is not always necessarily DNA, but indeed it can be RNA. Examples include PCR assay designated for the diagnosis of viral infections. In regards to these types of infections there usually a virus which has a RNA genome e.g. HIV, rabies etc. RNA cannot serve as a template for PCR, so for that reason reverse transcription is combined with PCR to make RNA into a complementary DNA suitable for the PCR template in order to be tested. To convert the RNA into DNA complements or cDNA inserts, the conversion can be carried out by simple enzymatic reaction with the enzyme reverse transcriptase. The DNA sample can then be amplified by PCR.
Solid phase PCR
Solid phase PCR allows very specific and sensitive detection of a target nucleic acid also called enzyme linked oligonucleotide sorbent assay, this is a solid phase sandwich hybridization assay, which is very close to the well-known procedure of ELISA.
Quantitative PCR (Q-PCR)
In molecular biology, real time polymerase chain reaction is a laboratory technique based on the PCR which us used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification of one or more specific sequences in a DNA sample. Quantitative PCR Is a PCR based method that is able to simultaneously amplify and detect changes in the amplicon concentration. Quantitative PCR collects data during the PCR amplification by utilizing fluorescence signal emitted by either special probes or DNA binding dyes (Dhanasekaran, Doherty and Kenneth, 2010).
Asymmetric PCR
In an asymmetric PCR, the reaction always amplifies one DNA strand in a double stranded DNA template, this makes it useful when the amplification of only one of the two complementary strands is taken into consideration because thee strands are needed in sequencing. Due to asymmetric PCR having a low reaction efficiency it is not widely used, it is also harder to optimize the primer ratios, amount of starting material and number of amplification cycles. During this assay the limiting lower concentration primer has a higher melting temperature than the higher concentration primer to maintain the efficiency of the reaction. Asymmetric PCR is similar to a normal PCR, the only difference is that the amount of primer for the targeted strand is much more than that of the non-targeted strand. As the method progresses the lower concentration limiting primer is quantitatively incorporated into newly synthesized double stranded DNA and then can be used.
Application of PCR:
PCR is used a lot in forensics, medical diagnostics where DNA samples are checked. A lot of medical applications include how the PCR is used in genetic testing procedures like genetic disease mutations, or detection of disease causing genes (Steffan, 1991). Forensic applications include the genetic fingerprinting method, where the PCR can be a tool to recognize one person in millions to solve a crime report or suspects for an investigation. It is also useful in paternity testing where DNA Is tested to match an individual results with that of their possible parents and children. PCR also plays a big role in the Molecular genetics field of research as it can help to compare two different organisms and their genomes, there are other phylogenetic analysis and gene expression analysis where the PCR can play a major role to aid in research and answers. PCR is highly used in genetic research, some of its applications are the study of gene expression patterns, rapid amplification of tiny DNA fragments using techniques like northern and southern blot hybridization, using DNA sequencing to study genetic mutations and their consequences. PCR also plays a role in virology where it can help to detect nucleic acids to understand the behavior of virus during an infection. PCR also allows the identification of microorganisms for the diagnosis of fungal infections in the study of mycology and parasitology (Lakshmi et al., 2011).
PCR method has been around since the 1980s, it is still used widely across the world for scientific and DNA research. Like every method there are certain advantages and disadvantages which can be spoken about. The major benefit of the PCR is that it only requires very minute amounts of DNA samples which are needed for amplification, the procedure makes copies of those minute samples and multiplies them largely in number, this takes place as the PCR is a sensitive procedure. Starting material used in small quantities which makes the procedure good to start off with. Secondly another good advantage of the PCR is that the oligonucleotide primers used can be used at low temperatures, this keeps the enzyme working thoroughly because the enzymes will denature at very high heat. If the temperature is kept high throughout the procedure this can be costly and it would make the procedure expensive to run. There are certain disadvantages that come with the method too like the sensitivity of the procedure can have an effect on the genetic material, this makes them less sensitive to anti-microbial data, giving unreliable results. This can lower the performance of the PCR as It only bound to specific data (Porter-Jordan and Garrett, 1990).
In conclusion we can say that polymerase chain reactions is a technique used worldwide in the molecular genetics industry which helps in the analysis of short sequence DNAs. PCR can help in answering many different medical questions and can be sued in the diagnosis of patients and treating them also. This is because PCR can actually detect the pathogenic organism in patients e.g. patients suffering from HIV or other virus related problems.
References:
Elder, R. H. (2016) Clinical & molecular genetics. University of salford , salford. Retrieved
Dhanasekaran, S., Doherty, T. and Kenneth, J. (2010). Comparison of different standards for real-time PCR-based absolute quantification. Journal of Immunological Methods, 354(1-2), pp.34-39.
Lakshmi, V., Sudha, T., Rakhi, D., Anilkumar, G. and Dandona, L. (2011). Application of Polymerase Chain Reaction to Detect HIV-1 DNA in Pools of Dried Blood Spots. Indian Journal of Microbiology, 51(2), pp.147-152.
Steffan, R. (1991). Polymerase Chain Reaction: Applications In Environmental Microbiology. Annual Review of Microbiology, 45(1), pp.137-161.
Valones, M., Guimarães, R., Brandão, L., Souza, P., Carvalho, A. and Crovela, S. (2009). Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Brazilian Journal of Microbiology, 40(1), pp.1-11.
Walters, S. (2012). Real-time PCR in Clinical Diagnostic Settings. Journal of Medical Microbiology & Diagnosis, 01(03).
INABA, H. (1989). Polymerase chain reaction (PCR). Blood & Vessel, 20(4), pp.365-367.
New Developments in Quantitative Real-time Polymerase Chain Reaction Technology. (2014). Current Issues in Molecular Biology.
Porter-Jordan, K. and Garrett, C. (1990). Source of contamination in polymerase chain reaction assay. The Lancet, 335(8699), p.1220.
Rogers, B. (2015). The Evolution of the Polymerase Chain Reaction to Diagnose Childhood Infections. Pediatric and Developmental Pathology, 18(6), pp.495-503.
Vögtlin, A., Bruckner, L. and Ottiger, H. (1999). Use of polymerase chain reaction (PCR) for the detection of vaccine contamination by infectious laryngotracheitis virus. Vaccine, 17(20-21), pp.2501-2506.