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Introduction
Polymerase Chain Reaction (PCR) is a very influential technique that helps in amplifying the specific segments of DNA. This biochemical procedure is performed in-vitro that is quite different from cloning. This technique is a fusion of synthesis protocol of distinct repeated segments of DNA and thermostable DNA polymerase application that has given scientists a very potent analytical, immunological and molecular technique used prevalently in clinical laboratory science (Garibyan & Avashia, 2013). PCR is based on three primary steps that include denaturation of the template into single strands, annealing of primers to each parent strand for synthesizing novel strands and the third step involves the extension of newly developed DNA strands from the primer sites. These procedures can be performed using any DNA polymerase that delivers the newly formed desired segments of original DNA sequence. To obtain multiple rounds of synthesis the templates are denatured repetitively along with the addition of fresh polymerase on each denaturation step (Delidow et al., 1993).
This process results in DNA amplification and it creates an opportunity for the measuring or detecting specific genes, which might be of great interest to a person (Shen, 2013). DNA comprises of a repetition of sequences in four bases; thymine, adenine, cytosine, and guanine. The sequences result in the formation of two strands, which are joined by a double helix structure that is made of the hydrogen bond. As such, each half of the helix exists as a complement to the other. Human beings have difference sequences for the bases found on the DNA strand, which ensures that each individual has a unique genetic makeup. The bases arrangement in each gene helps in the production of the RNA, which further produces protein. PCR is performed in several cycles using thermocycler. Thermocycler has the ability to decrease and increase specimen temperatures in defined intervals (Shen, 2013). The first step is the separation of the DNA strands, followed by the cooling of the sample and attaching the primers. Heat is then applied to each of the strands, which results in the duplication of the strands. The cycle is repeated and after 30 or more cycles, billion of original DNA section copies are obtained, which can be subjected to molecular diagnostic tests (figure 1) (Shen, 2013).
PCR is the most powerful laboratory technique ever developed. The relatively low-cost, specificity and sensitivity of this procedure provide it a unique combination of flexibility that revolutionized the genetics. It has opened the doors to the various hidden aspects of genetics. There are various modifications that were done to PCR techniques over a period (Valones et al., 2009). The kinetic or real-time PCR has provided a new dimension to this technique especially in the probe and primer designing as well as the advancement of experimental conditions (Innis et al., 2012). The rapidity and the comparative flexibility of current PCR techniques facilitate the acquisition of quantitative data that is impressive, reliable and can be integrated for objective analysis. The discovery of PCR has been valuable for diverse biological developments such as gene expressions in a recombinant system, molecular-genetic analysis, genome sequencing, and rapid determination of parental origin and diagnosis of infectious diseases (Innis et al., 2012). PCR technique is widely used in the diagnosis of the diseases for identifying viruses and bacteria, and matching of criminals to the various crime scenes (Shen, 2013). However, such may have an error that originates from PCR fragments mutations but PCR demonstrate excellent detection limits. The latest innovation of PCR known as real-time PCR (RT-PCR) is widely accepted by clinical diagnostics because of its high competence of producing quantitative results. RT- PCR produces more accurate and faster result than conventional PCR that was only able to deliver qualitative results (Valones et al., 2009).
History of PCR Technology
The PCR technology was firstly developed by Kary Mullis and co-worker in early 1980’s at Cetus Corporation, California. This method was firstly presented at the American Society of Human Genetics Conference in 1985, October (Bartlett & Stirling, 2003). The first application of PCR was also published in the similar year that was an analysis of sickle cell anemia. In the early form the PCR was very time taking and intensive, but over the time, it has improved and now developed as a fast and reliable method. For this research, Kary Mullis received Nobel Prize in Chemistry in 1994 (Mullis et al., 1986). The original concept of the PCR was based on the fusion of several techniques that were already present at that time. The development of short length single stranded DNA (Oligonucleotides) was common at that time. Moreover, the integration of these Oligonucleotides to the target-specific progression of novel DNA copies with the help of DNA polymerases was also a common tool used by the biologists at that time. Polymerases were already being used in various biological applications. Kary Mullis just fused these two concepts by juxtaposing the two Oligonucleotides of DNA in a complementary manner to opposite strands. This approach facilitated the specific amplification of the region between two DNA segments and to obtain this, repetitive polymerase enzyme was added to continue a chain reaction. This Mullis work was inspired by Khorana’s work in the late 1960s and 1970s that were based on three simple steps highly similar to current PCR that are annealing of primers and template extension, separation of newly developed strands from the template and re-annealing of the primer. The DNA polymerase applied in PCR techniques was taken from E. Coli. Although DNA polymerase enzyme was highly significant for an extensive range of biological applications but it couldn’t be successful in PCR because the heating on a high temperature to denature the double-stranded DNA during PCR also used to inactivate the E. Coli-DNA polymerase. Thus, for this purpose, a more stable enzyme was required that could remain intact at 95o C. This problem was resolved by the discovery of Thermophylus aquaticus that was the organism of extreme-hot springs and also produces a heat-stable form of DNA polymerase. Gelfands and his colleagues at Cetus cloned this enzyme that enables a complete PCR procedure without opening the reaction tube time after time (Bartlett & Stirling, 2003; Templeton, 1992).
The PCR Reaction Components
The main components of PCR reactions are DNA template, DNA polymerase enzymes, primers, and nucleotides. DNA polymerase enzyme needs a primer while processing because DNA polymerase enzyme can attach a nucleotide only to a 3-OH group. In this case, the primer helps in attaching the nucleotide first to itself. This enzyme can formulate the novel strands of DNA that are complementary to the target nucleotide sequence. The enzyme Thermis aquaticus, Taq-DNA polymerase is mostly used in PCR reactions. Pfu-DNA polymerase enzyme is the form of this enzyme which is extracted from Pyrococcus furiosus is extensively used due to its high fidelity while copying DNA. The two distinct characteristics associated with these two types of DNA polymerase are that they can produce two strands of DNA with the primers and DNA templates. Secondly, they both can bear high temperatures (Polymerase Chain Reaction, 2016).
DNA templates are a sample of DNA pieces that holds the target nucleotide sequences while initiating the reaction a high temperature of 95oC is applied to denature the original double stranded DNA molecule into a single-stranded DNA segment. These templates serve as a primer to commence the reaction. One DNA molecule is applied to produce to deliver 2 copies and then 4 copies in an exponential manner. DNA polymerase enzymes facilitate doubling of single strands into long molecular strands using DNA building blocks that are Adenine (A), Cytosine (C), Thymine (T), and Guanine (G). The short segments of DNA, primers attach the building blocks to make a longer DNA molecule from its end. Thus template, primers and nucleotides (building blocks) mutually construct the similar copies of the template in the presence of a polymerase. After denaturation of DNA, the temperature is reduced to around 50 oC to enable primers to attach themselves with the target genes. A suitable temperature for polymerase activity is 72 oC that fastens its activity. Now, this course of temperature is repeated for every cycle until the desired copied of target DNA is achieved. The PCR reaction produces target DNA segments in an exponential manner Polymerase Chain Reaction, 2016).
Parameters that Impact the PCR Process
Thermo-stable DNA polymerase
For a flexible and cost effective production of PCR, a thermostable DNA polymerase enzyme is an essential component that enables the catalysis in a template-dependent manner to synthesize large DNA products. The most popular polymerase used in PCR is TAQ-Polymerase that shows a specific activity of approximately 80,000 units per protein mg. The estimated efficiency of primer extension in the presence of TAQ-polymerase is 0.7, thus, when it reaches to 1.4x1012 to 7x1012 amplified molecules the enzyme becomes limiting (The Polymerase Chain Reaction, 2011).
Deoxynucleoside Triphosphate (dNTPs) or Building Blocks
A standard PCR reaction employs an equimolar quantity of all four bases or dNTPs. In a TAQ-polymerase PCR reaction, the recommended quantity of each dNTP is 200-250 µM of each dNTP along with 1.5 mm Magnesium Chloride (MgCl2). dNTPs in high concentrations are prohibited to avoid sequestering of Magnesium (The Polymerase Chain Reaction, 2011).
Pair of Oligonucleotides for DNA Priming
Oligonucleotide primers are needed to design carefully to produce efficient and high yield. It also helps in preventing the amplification of undesired sequences. To design the Oligonucleotide sometimes several segments of target DNA are amplified in such cases an amplification reaction is run that involves multiple pairs of primers in the reaction mixture. Based on the literature search it is found that a standard reaction involves 0.12 to 0.5 µM of each primer. This extent of primers is suggested to be sufficient for 30 amplification cycles for a 1 KB DNA segment (The Polymerase Chain Reaction, 2011).
Template DNA
The quantity and quality of template DNA that consists of target DNA sequence is a very significant component of PCR. It should be in highly pure form and can be integrated to PCR in any single or double stranded form (Mullis & Faloona, 1987). Linear DNAs can be amplified more effectively in comparison of closed and circular templates. The template DNA should contain target sequence where the source and the method play a critical role. It is suggested to use genomic DNA for efficient amplification in comparison of plasmid DNA because genomic DNA contains one copy of target DNA per genome while plasmid DNA are comparatively small and enriched with specific target sequences. According to experts, the maximum of 100-ng genomic DNA is sufficient for PCR amplification depending upon genome background (Polymerase Chain Reaction, Vlab.amrita.edu). In the case of the mammalian genome, it depicts 10,000 genomes comparable in the reaction (The Polymerase Chain Reaction, 2011).
PCR Reaction Buffer
The next component of PCR reaction is its buffer to maintain the pH. The recommended pH for PCR protocol is 8.32 to 8.88, which is added with the commercial polymerase at a concentration of 10 mM (10x). Most of the commercial buffers contain Magnesium Chloride that provides a Mg++ Cation to the Type-II enzymes as a cofactor. These Type-II enzymes present enzymes such as restriction endonucleases and polymerases. Tris-Cl is also used for the reagent base that under incubation condition at a 72 oC produces a buffer pH of 7.2. The purpose of adding reaction buffer is to achieve an ideal pH and to create a monovalent salt milieu for the final volume (Polymerase Chain Reaction, Vlab.amrita.edu). The standard concentration of buffer reagent is suggested as 1.5 mM which can be modified according to the optimization of the PCR reaction (The Polymerase Chain Reaction, 2011).
Monovalent and Divalent Cations
Most of the thermostable DNA polymerase enzymes need Cations such as Mg++ for its activity. Mn2+ and Ca2+ are found less effective for PCRs. Due to the binding of DNTPs with Mg2+, it is recommended that the molar concentration of Cations should be higher than the phosphate groups in the environment. The standard recommended the concentration of Cation is 1.5 mM that is also adjustable according to the required conditions. Several studies have shown that increasing the Mg2+ concentration to 4.5 mM may also result in reduced or elevated non-specific priming. Thus, the concentration of the Cation Mg2+ should be empirically determined for every template and primer blend. It is also suggested that the DNA template preparations should be void of chelating agents like Anions because they can sequester Cations.
Standard PCR Buffer Reagents consists of 50 mM KCl that is sufficient for the amplification of an approximately 500 BP DNA segments. Increasing the monovalent K+ concentration can increase the production of shorter DNA segments (Polymerase Chain Reaction, Vlab.amrita.edu).
PCR Protocols
Figure 1: PCR protocol (Polymerase Chain Reaction, 2016).
PCR is a repetitive process with the three main segments, first is denaturation which is followed by annealing and extension of annealed primers (figure 1) (Erlich, 1989).
Denaturation
In the denaturation step mainly two things occur first the target DNA segment denature to single strands and secondly heat generates convection currents that trigger Brownian motions in the mixture molecules. Higher the proportion of G+C content in the DNA template greater the temperature is needed to separate the DNA strands. The time requirement also increases with the length of DNA molecules. In the presence of low temperature and less time only AT segments are denatured. When the temperature is decreased in the later stages of PCR the template DNA reanneals into its original form. The standard observed time is 45 seconds sufficient to denature Linear DNA segments containing more than half G+C content at the temperature of 94-95 oC (figure 2) (Mullis & Faloona, 1987).
Figure 2: A common three-step PCR cycle (The Polymerase Chain Reaction, 2011).
Annealing of Primers
The temperature of the mixture decreases with the termination of denaturation as it proceeds to annealing phase. During this phase, the primers go through random temperature zones until they reach a particular Tm for the primers where they can find their perfect complement for annealing (figure 2). This temperature further reaches a temperature, Ta at which maximum annealing takes place. Too low-temperature results in nonspecific annealing while high-temperature results in poor oligonucleotide annealing. In the initial phase only target DNA segments act as templates for primer annealing but in the later stage when process advances each one primer attaches itself as complement to these target DNAs and polymerase facilitates this process with extension without any terminating point until the denaturation temperature reaches to a level where it commences new cycle (figure 3) (Polymerase Chain Reaction, 2011).
Figure 3: The first four cycle of PCR (The Polymerase Chain Reaction, 2011).
These PCR products are joined from the only one end and known as semi-bounded or anchored DNAs. In the second cycle these anchored DNAs also act as a template alike original target DNAs. The anchored DNAs form anchored products in each cycle whereas the original DNAs bind with the complementary primers, thus producing primary distinct PCR amplicon. Later on, along with the anchored DNAs and template DNAs, the newly formed amplicons start serving as targets for PCR priming. Onward third cycle DNA segments amplify geometrically while longer amplicons build up arithmetically. It is estimated that Taq polymerase can polymerize around 2000 nucleotides per minute at the temperature of 72-78 oC where extension takes one minute for a product of 1000 bp. Often experts prefer three times longer extension period for the final cycle to provide sufficient time for completion of all amplicons (Polymerase Chain Reaction, 2011).
Application of PCR in Clinical Biology
Virology
Contemporary advancements in molecular biology and PCR have facilitated the depiction and detection of viral nucleic acids enabling the magnification of the desired and precise section of the genetic material (Valones et al., 2009). PCR helps in analyzing the gene sequences that assists in identifying the holistic characterizations of virus particles, its genotype, subtype, mutations, variation and resistance such as HIV, HCV (Human Cytomegalovirus), HBV (Hepatitis B virus) etc. PCR contributes in epidemiological investigations because of its ability to enumerate nucleic acids in a fast and single reaction (Gouvea et al., 1990).
Mycology and Parasitology
The unique capability of PCR to quantitatively and accurately identify the microbes is highly valuable to fungal epidemiology. In the field of phytopathology, the before time detection of infectious agents helps in early identification of pathogens as well as in early disease control measures. Through latest RT-PCR method, the samples can be directly tested without isolation. PCR is very fast and extremely explicit and can even sense trace amounts of fungal DNA in the sample (Bretagne, 2003).
Microbiology and Bacteriology
PCR, especially RT-PCR has immediate and significant connotation into the diagnostic clinical microbiology. The improved sensitivity, flexibility and quick response has made it more convenient and attractive option for microorganism detection in human. The detection of anaerobic bacteria had been a challenge several decades back which are a major factor for mortality and morbidity among humans through an extensive range of infections. PCR has become a powerful tool of understanding the parasite-host relationship that reveals the taxonomic position of the pathogens. PCR is used is identifying various bacteria such as PCR for Gadnerella vaginallis, genital tract bacteria, Chlamydia Trachomatis, Lactobacillus, Micobacterium tuberculosis, Neisseria gonorrheae, Bordetella pertussis and Mycoplasmas hominis.
Dentistry
PCR has been an excellent mean of detecting the periodontal pathogens in the subgingival samples. Due to the sensitivity and rapidity of PCR it could be observed that various pathogens are involved in periodontal disease through the crevicular fluid such as Human Cytomegalovirus (HCMV), Herpes Simples Virus (HSV), Epstein-Barr Virus Type I and II (EBV 1 and 2), Human Papillomavirus (HPV) and Human Immunodeficiency Virus (HIV) (Santos et al., 2004).
Conclusion
PCR has been a very potent technique that has revolutionized the different field of molecular biology and pathology. Over the time various advancements have taken place in the selection of PCR components and protocols but it is emerging as a more versatile, flexible and cost-effective technique. In clinical laboratory science PCR has expanded its broad hands and assists in basic to critical cases from as epidemiological problem to a high-profile criminal case..
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