Polymerase Chain reaction (PCR)
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Abstract
Polymerase chain reaction (PCR) is greatly used in molecular genetics. It entails amplification of a single DNA strand into millions of similar DNA fragments. It involves three stages in each cycle. It is repeated to about 30 cycles. This method is vital as it is used in various processes such molecular identification, genetic engineering, and sequencing. The three stages in each cycle have varying duration and temperature. A thermal cycler is involved in the regulation of temperature in various stages. Over time, various modifications have been done to PCR technique so that it can be applied in specific roles. The PCR has been of aid in the diagnosis of diseases and other numerous applications. In the near future, PCR will be advanced and perhaps replaced by better techniques. Nevertheless, PCR will remain critical for future advancements in molecular genetics.

Introduction

Polymerase chain reaction (PCR) is broadly employed by scientists in biochemistry and molecular biology. Thus, its essence cannot be underestimated in the development of genetic analysis and gene manipulation. The technique was established by Karry Mullis in the early 1980s. It entails amplification of a single or several DNA fragments into millions of identical copies of DNA. The process is done by repeated number of cycles that range between 30 to 40. This results into a chain reaction. The automated machine used in the cycling process is a thermal cycler. It regulates the temperature in various stages by frequent elevation and lowering of temperature depending on the PCR stage. (Verkuil, Belkum & Hays, 2008). The necessary materials required in PCR technique include DNA templates that contain the target sequence that is amplified, DNA polymerase, primers and deoxynucleotide triphosphates (dNTPs).

There are various DNA polymerases that can be utilized in the PCR process. Pfu DNA polymerase that is extracted from Pyrococcus furiosus and Taq polymerase derived from bacteria Thermis aquaticus are commonly used. These DNA polymerases are stable at high temperatures; thus, they are not likely to be denatured compared to Escherichia coli DNA polymerase that was used initially. E. coli DNA polymerase has an optimal temperature of 37oc thus it is labile at high temperatures. Its utilization forced addition of more E. coli polymerase at the beginning of each cycle. However, introduction of the latter thermal stable DNA polymerases simplified and increased the efficiency of PCR amplification due to enhanced specificity of the DNA target. This process consists of three stages namely; denaturation, annealing, and extension.
The temperature applied in each stage varies. The PCR technique has many vital applications that are worth mentioning. Polymerase chain reaction has extensively been used in DNA fingerprinting, mutation, genetic correspondence, diagnosis of genetical disorders, grouping of organisms, pre-natal screening and drug discovery and development. Furthermore, it is also used in forensic science to apprehend criminals as well as in paternity or maternity tests.

Steps carried out in polymerase chain reaction.
There are three stages taken during polymerase chain reaction in each cycle. They are redone up to about 30 cycles. They involve temperature change and different duration of each stage. To elaborate further, the temperature in these stages is varied depending on the quantity of divalent ions, DNA polymerase and divalent ions used. Some DNA polymerase used require high temperature of up to 98oC so as to be activated thus this brings the variance. In addition, primers melt at different points depending on the ratio between cytosine- guanine bonds to adenine- thymine bonds. Cytosine guanine bonds are strong since they involve three hydrogen bonds while the latter has two hydrogen bonds. Hence, primers with a higher ratio of cytosine-guanine to adenine- thymine ratio require more energy to break them up.

The following stages are involved

1) Initialization

This stage is done to DNA polymerases that require to be activated by temperature elevation. It is essentially carried out by utilization of hot-start PCR in polymerases that need high temperature for activation. Hot start PCR is an advanced type of PCR that inactivates DNA polymerase at lower temperatures so as to avoid non-particular amplification of DNA target sequence. It is done at a temperature of between 94 to 98oC for about one to nine minutes.

2) Denaturing.
The goal is to obtain single-stranded DNA template that acts as the starting material for amplification of target site fragments. It does so by introduction of heat of about 94 - 98oC in the thermal cycler for about thirty seconds. High temperatures break the hydrogen bonds formed between the various corresponding nucleotide bases that join the two strands thus forming single DNA strands.
3) Annealing.
The annealing step requires a decreased temperature so as to ensure that the primer binds to the corresponding target sequence. However, it should be a bit higher so as to ensure the annealing of primers is specific. The temperature applied at this stage is about 55oC to 60o C. This temperature is about 5oC less the primers melting point. Extremely strong hydrogen bonds are formed when primers bind to a specific matching nucleotide bases. This phase is carried out for about 20 seconds. After which DNA polymerase begins elongation the target DNA sequence by use of various deoxynucleotide triphosphates.

4) Elongation of the target DNA nucleotide sequence.
The elongation step relies on DNA polymerase. The temperature employed at this stage is dependent on DNA polymerase optimum working condition. Taq polymerase that is mostly used it functions optimally at 76 – 80oC. Hence, a temperature of 73oC is used for optimum enzyme activity. It elongates the new strand by addition of deoxynucleotide triphosphates that correspond to nucleotide bases in the DNA template in the 5` - 3` direction. It is achieved by condensation of the 5 - phosphate group present on the nucleotide base with the 3` hydroxyl group present on the DNA template nucleotide bases. It leads to the formation of hydrogen bonds between the template and the newly elongated target sequence strand. The elongation duration depends on the size of target DNA sequence and the enzymes efficiency. However, under optimum condition each elongation step leads to doubling of DNA templates thus bringing an exponential increase in DNA target sequence.

5) Ultimate elongation.
The key aim of this step is to ensure that there is no single-stranded DNA left out without being extended. It is done after the final polymerase chain reaction cycle. It is carried out for about 10-12 minutes at a temperature of about 73 - 740C
6) Final hold
It is done for temporary storage of the reaction. It is carried for an unspecified duration of time at 5- 14oC
After the overall PCR process is completed, the amplification the target DNA sequence is confirmed by the use of agarose gel electrophoresis. The agarose gel concept entails utilization of a DNA ladder that has fragments of varying. The polymerase chain reaction products are separated based on their various sizes and matched beside the ladder so as to determine whether the target strand was amplified.

It is a visualized image of agarose gel electrophoresis. Use of Ethidium bromide visualizes it. It enables determining the size of various DNA products present in the PCR.

L – It represents the DNA ladder which has about 1000 kilobase pairs of DNA.

1-10 – it represents varying sizes of DNA fragments that were products of PCR whose sizes are determined based on the Ladder.

The determination of the various sizes of polymerase chain reaction products helps in concluding if the target DNA fragments were amplified optimally.

Optimization of PCR
A times PCR technique may be unsuccessful due to various reasons. It is due to increased sensitivity of the reaction. Hence, it makes it delicate to impurities that may be introduced during various stages of PCR. It reduces the specificity of primers to the target sequence. Therefore, to ensure optimal efficiency and specificity a number of techniques have been introduced.

1) Modification of the buffer concentration
Tris 10 mM and potassium chloride is contained in the majority of the buffers. The binding of the primer is enhanced by KCl. However, when the concentration of KCl is more than 50 mM it deters DNA polymerase functioning. Hence, use of buffers at certain concentrations optimizes the overall PCR process. In addition, other buffers such as glycerol, gelatin, Triton X-100, BSA, Tween 20 and DMSO can be utilized to increase specificity of primers. However, altering their concentration affect the activity of Taq polymerase also.

2) Regulation of the cycling requirements.
In the denaturation stage, Taq polymerase requires to be activated by use of Hot- start PCR. A temperature of around 94oC is needed for this. It ensures that DNA polymerase activity is initiated so that it can work. The temperature used in the annealing stage is less 5oC the melting point of the primer so as to avoid its destruction. The increase in temperature in the annealing stage decreases yield while an increase in temperature diminishes specificity. Hence, the optimal temperature of 55oC is applied.

3) The number of cycles of PCR
Theoretically the produce of DNA amplification increases exponentially. The number of DNA fragments produced is given in the following formula as 2n = number of target DNA sequence. The number of cycles is represented by letter n. The number of cycles carried out in PCR is usually 20 – 30 cycles. Taq polymerase has half-life of about 30 minutes at 95oC. Every PCR cycle has a denaturation stage that employs 94oC temperature for one minute. Hence, in 30 cycles there is 30 minutes exposure of polymerase enzyme to 94oC. (Shafique, 2012). At the 30th cycle, the Taq polymerase is half the original amount. It reduces the yield of PCR progressively as the number of cycles are increased. Therefore, it is important to use only a maximum of 30 cycles in each PCR process.

4) Magnesium chloride utilization in primer binding

Magnesium chloride is essential in annealing of the primer. In addition, it affects the melting point of the DNA template, dNTPs, specificity in the annealing of primers and DNA template. Finally, it affects enzyme activity and specificity. An increased concentration brings about reduced specificity while low levels of magnesium decreases the overall yield. Hence, magnesium concentration should be higher than dNTPs.
5) Designing of the primer.
The primer size used should not be more than 30 nucleotide sequences while the C and G content should be 40 – 60 % of the total bases. It enables breakage of the formed double strand in the subsequent Denaturation step. While at the 3` ends should have a CG, GC or C or so as to ensure the formation of strong bonds.

Modifications of PCR technique.
The modifications of PCR have been done progressively since its discovery. It has significantly enabled its use in various processes such as the analysis of gene mutation. The modifications include
Quantitative- real-time PCR technique, the aim of this PCR is to determine the amount of target DNA or RNA sequence amplified. (Cohn, Russell, 2012). It has specialized thermal cycler that measure the quantity of products yielded from the amplification in the exponential phase. It entails the utilization of fluorescent dyes in quantifying the amount of amplified DNA or RNA.

Multiplex analysis - This uses many primers that have different DNA targets. Thus, it is possible for amplification of two or more targets in a single reaction. This technique has been involved in genetical analysis so as to detect gene mutation and genetic variations among individuals.
Asymmetric PCR - This method of PCR uses only one primer in the replication of the DNA. However, it may use two primers, in which one primer is applied in limited amounts in the thermal cycler. After the limited primer becomes depleted, the annealing of the other excess primer increases progressively. The aim of this type of PCR is to produce a single-stranded DNA. It is commonly used in sequencing of DNA strands.

Nest PCR - The aim of this technique is to optimize specificity in the annealing stage and binding of dNTPs to the target strand. It is carried out by use of two pairs of primers involving two stages. The first set of primer amplifies the DNA yielding products. The amplified products are used in the subsequent step utilizing one or two primers that have their binding sites in the initial set of primers. It substantially increases specificity.

Variable number of Tandems repeats (VNTRs) PCR - This technique of PCR focuses on the regions that exhibit differences in length. Use of gel electrophoresis determines the sizes of PCR. This method is widely employed in DNA fingerprinting.
Polymerase cycling process PCR - This method involves synthesis of long strands of DNA from many small fragments that overlap.
Touchdown PCR technique - it was introduced with the aim of increasing efficiency in the replication process. Usually, it has an elevated temperature during the initial cycles of the PCR. The temperature is about 3oC higher than the melting point of primers. The temperature is then decreased in final stages. It is carried out so as to increase specificity of primers in the annealing step.
Applications of PCR method
1) Forensic science – In most criminal cases only tiny amounts of DNA are obtainable as evidence. Hence, forensic scientists use PCR to amplify the small fragments of DNA into thousands of DNA fragments. It is used for linking suspects to a particular crime.
2) Diagnosis of diseases- It has been used in the diagnosis of infectious diseases. It enables detection of viruses, bacteria, and mycobacteria through tissue culture array. It makes it possible to differentiate between pathogenic and non-pathogenic genes.
In addition, it has also been used in the diagnosis of various types of cancer such as lymphomas and leukemia. It is achieved by use of PCR arrays that analyze various malignant genes or sites of transformation in cells.
It helps in the diagnosis of Alzheimer diseases - The PCR array enables identification of 84 genes that are responsible for the start and progression of the diseases. It is made possible by use of real-time PCR technique. The genes involved include those that lead to inflammation and neurotoxicity.
3) Analyzing enzymes and transport carriers involved in drug metabolism

About 84 genes are involved in the metabolism of hormones, drugs, various nutrients, and toxins. (Bustin, Nolan, 2013). In cases of alteration of genes responsible for metabolism various diseases of metabolism results. Genes that code enzymes that are involved in the metabolism such as transferases and cytochrome p450 are analyzed by use of PCR so as to identify if there are abnormalities and how they function.

4) DNA cloning and hybridization probes - These processes require many DNA nucleotides. Thus, they are amplified by into millions of DNA that enables the cloning and hybridization to be successful.

5) DNA sequencing – This helps in determining the DNA pattern of unknown genes.

The future of PCR technique.
PCR technique has developed progressively since its discovery by Karry Mullis. It has significantly improved the overall genetic screening and analysis in molecular biology. Nevertheless, it has been faced with various shortcomings such as non-specificity. The reduced cost of the PCR in the near future is going to attract many enterprises into the market. It will lead to advancement of technology in mainly environmental and diagnostic fields. The technology and development of science techniques are taking place daily. Hence, as time goes by, the PCR technique is becoming outdated. In the coming times, advancement in the PCR technology will have gone a notch higher.
Conclusion
In conclusion, PCR has remained vital in many processes of molecular biology. Its application in various fields has led to better understanding of molecular genetics and substantially helped in providing better medical services. Indeed, PCR will remain elemental for further advancement in genetics and molecular biology.

References
1) Verkuil, E., Belkum, A., & Hays, J. (2008). Principles and Technical Aspects of
PCR Amplification (1st ed., p. 330). Springer.
2) Cohn, R., & Russell, J. (2012). Real-Time Polymerase Chain Reaction (p. 88). Springer.
3) Bustin, S., & Nolan, T. (2013). PCR Technology: Current Innovations (3rd ed.). CRC press.
4) Shafique, S. (2012). Polymerase Chain Reaction: Procedure, Principles, Real time
PCR, Optimization, Applications, PCR Arrays, Array System Performance, Protocol,Variations. LAP LAMBERT Academic Publishing.
5) Verma, S., Kennath, S., & Sennath, S. (2012). Polymerase Chain Reaction: Expedition Of A
Ubiquitous Tool (p. 132). LAP LAMBERT Academic Publishing.