Polymerase Chain Reaction (PCR): The New Gold Standard!

Polymerase Chain Reaction (PCR) represents a laboratory methodology that concentrates rapidly in making billions of a specific region of DNA samples allowing scientists related to this field to use a microscopic DNA sample and amplify it to a more considerable amount necessary for experimental research study hence the name” molecular photocopying.”

The emergence of Polymerase Chain Reaction continues to transfigure biological science since the initial time, allowing more and large amounts of DNA to be effectively used in scientific projects such as the Human Genome Project (Mullis, 1990). It remains to be considered among the best breakthrough in molecular biology, making its inventor, Kary B. Mullis, awarded the Nobel Prize for Chemistry in 1993, an honour that many scientists treasured around the globe.

Driven by its ultimate goal, new current developments in PCR allows scientists to carry out variations in experiments they are interested in, such as gene behaviours developments or genetic compositions that has proved to be beneficial in the forensic field in terms of crime detections and analyses of criminals features. PCR has proved to be an integral factor, especially in medicine and biological fields.

The knowledge continues to be applied by clinicians and researchers to detect diseases, cloning, paternity testing, mutation identification, microarrays, and gene sequencing, contributing significantly to the many applications of PCR.  Detection of pathogens through DNA analysis, bacteria identifications, and antigen-antibody interactions remain one of the most preeminent medical applications of the modern Polymerase Chain Reaction.

Figure 1: Mouse genotyping tips (Thermofisher)

Existing limitations of Polymerase Chain Reactions limit its full potential of transforming the medical and biological areas. PCR is only applicable in the identification or absence of a known gene or pathogen, and the same applies to its DNA polymerases that are prone to mistakes leading to mutations in the Polymerase Chain Reaction structure.

Another known limitation falls on primers used in Polymerase Chain Reaction as they often cause non-specific binding to sequences that are similar but less identical to the subject sample DNA. Since it is applicable almost universally, it is vital to grasp and understand the types, principles, procedures, applications, machines used, and the various scientific accomplishments and breakthroughs in Polymerase Chain Reaction, as discussed in this article.

Types of Polymerase Chain Reaction

The ambidexterity of PCR over time constitutes too many PCR variants that often allow some modifications to be applied to the standard PCR to realize the ultimate goal of the experiments. Different types of Polymerase Chain Reaction are highlighted below;

Amplified fragment length polymorphism (AFLP) PCR

It majorly utilizes a specific section of the digested DNA fragments to produce unique and distinctive fingerprints for the study’s genomes without knowing the genomic sequence. Through the usage of restricted enzymes, genomic DNA is absorbed, allowing the adaptors to attach themselves to the sticky regions of the fragments, which are later amplified by the selected primers compatible with the adaptor sequence.

According to size, precise visualization, and separation of the amplified sequences of the DNA fragments according to size are achieved.  AFLP continues to provide necessary knowledge on the genetic diversity among different species and the generation of genetic maps to understand molecular biology.

Figure 2: AFLP Illustration

Allele-specific PCR (AS-PCR)

It is also known as refractory amplification mutation (ARMS-PCR) and majorly focuses on the general analysis of single nucleotide polymorphisms by incorporating different primers and alleles. Both the mutant and normal alleles are amplified by the primers, leading to the single allele’s general amplification. Many areas in single gene point mutations, such as direct determination of ABO blood group genotypes, thalassemia, and sickle cell anaemia, continue to benefit from the AS-PCR.

Alu PCR

It is a biological approach that concentrates on the DNA fingerprinting approach focusing on various genomic loci in the Alu repetitive structure, allowing for recognizing genetic polymorphisms and mutations in human and primate genomes. The presence of Alu elements in the human genome enables scientists to associate them with the evolving role and as genetic markers in their study.  Genetically inherited human diseases and cancer continue to benefit widely in terms of their identification and detection through this PCR and understanding their mutations.

Polymerase Cycling Assembly

It refers to the synthesis of long DNA strands by conducting PCR of the long oligonucleotides and the short overlapping segments leading to the assemblage of the DNA in one structure. Polymerase enzyme facilitates significant hybridization leading to important usage of this type of PCR in molecular biology.  Assembly PCR helps produce large amounts of RNA and the required protein yield that are important in the biochemical studies.

Asymmetric PCR 

Generally associated with the process of preferentially amplify a specific DNA strand in a double-stranded DNA pattern. It is currently being applied in sequencing and hybridization probing, where the amplification of a single of the two complementary strands is necessary.

 Cold PCR

It refers to a unique type of PCR specializing in upgrading variant alleles from the existing composition of wild type and mutation – containing DNA irrespective of the mutation categorization. Cold PCR contributes to identifying mutations in oncology specimens, more so in heterogeneous tumours and body fluids. Patients recovering from surgery or chemotherapy continue to benefit from Cold PCR knowledge as the residual disease can easily be estimated.

Colony PCR

It refers to the method of evaluating and designing the DNA understudy by inserting it into the plasmid enriched with bacterial colony together with the appropriate DNA primers.  This PCR type continues to be widely used incorrect ligation identification and understanding the inserted DNA behaviour into the bacteria.

Conventional or Standard PCR

It is a primary type of PCR that mostly brings qualitative outcomes during the process of visualizing and detecting the DNA strands replications. The variant is applied in forensic studies analysis and infectious disease diagnosis.

In Situ PCR 

PCR takes place in cells or in fixed tissue on a slide leading to preservation of the cell morphology, which is then supported by applying proteolytic enzymes, providing a mechanism for the PCR reagents to integrate on the specific DNA.  This type of PRC continues to be applied in the study of embryogenesis and organogenesis.

Other Types of Polymerase Chain Reactions Include; 

  • Intersequence specific (ISS) PCR
  • Inverse PCR
  • LATE (Linear-After-The-Exponential) PCR
  • Ligation mediated PCR
  • Long-Range PCR
  • Methylation-specific PCR (MSP)
  • Miniprimer PCR
  • Multiplex PCR
  • Nested PCR
  • Real-Time PCR (Quantitative PCR (qPCR))
  • Variable Number of Tandem Repeats (VNTR) PCR
  • Touch down PCR
  • Thermal asymmetric interlaced PCR (TAIL-PCR)
  • Suicide PCR
  • Solid Phase PCR
  • Single Specific Primer PCR
  • RNase H-dependent PCR

Principle and Procedure of Polymerase Chain Reaction

PCR is an in vitro technique process is guided by the general principle of DNA polymerization reaction that uses DNA polymerase, primer sequence, and dNTPs taking place in various repeated cycles of heating and cooling in the thermal cycling process.  This principle allows as many as a billion times amplification of Specific DNA sequence and the applied methods supporting up to 10 and 40-kilo base pairs (kb) of DNA fragments amplification. The enzyme DNA polymerase assists in linking the nucleotides to the three ends of an oligonucleotide that is annealed to a longer template DNA allowing the enzyme to use it as a primer in the process of generating an elongated region of double-stranded DNA.

For the above principle and procedure to be successful various components and reagents are used. Microfuge tube, thermal cylinder, DNA template, Primer, Tris-Hcl, Mgcl2, Kcl, Gelatin or bovine serum, distilled water, Deoxyneuclotide triphosphates, and DNA polymerase presents the components necessary in the procedure of PCR. The recognized polymerase chain reaction procedure entails 20-40 constant cycles that constitute different temperature changes in each cycle.

Various steps/ procedure of Polymerase chain Reaction:

1 Initialization step.

It is the first step in PCR’s procedure consisting of a rise in temperature from 94°C to 96 or 98°C, more specifically when thermostable polymerases are applied for a range of 1-9 minutes. The main aim of this step is to ensure full activation of DNA polymerase that is used in the reaction.

2. Denaturation step.

The reaction is subjected to intensive heating ranging from 94-98 degrees centigrade for about 20-30 seconds. Hydrogen bonds forming strong bases are broken down during this step leading to high yielding of single-stranded DNA molecules

3. Annealing step.

Cooling of the mixture occurs at this step through an approximate temperature of 50–65 degrees centigrade for about 20-40 seconds making the primers anneal to the single-stranded DNA template. Formation of stable DNA-DNA hydrogen bonds are often formed in this step but dramatically depends on the primer sequence relationship with the template DNA sequence that lasts for about one minute in the cycle. The concentration of the primer in this process is very high compared to that of the template DNA leading to the formation of primer-template hybrid over the template strands’ re-annealing.

4. Extension/elongation step.

This process dramatically depends on DNA polymerase as it plays a crucial role in synthesizing and amplifying new strands of DNA in the reaction process. Depending on the specific type of the enzyme used, the temperature is bound to vary. Standard Taq polymerase works best at its optimum activity temperature at 75-80 degrees centigrade, assisting in the synthesis of complementary strands through the 3′-OH of the primer. The primers’ closeness allows the DNA segment to replicate since the applied primers complement the 3′-ends of the segment to be amplified.

Primer extension generally takes place at 72°C for about 2 minutes of the duration where the enzyme will disintegrate the available primers generating a new DNA strand making the DNA double-stranded again.  At regular instances, 2 Kb of DNA fragments are amplified, but special conditions need to be implemented to amplify longer segments. At optimum temperature, the DNA polymerase often yields a thousand bases per minute, causing an exponential amplification of the study DNA fragment.

5. Final elongation.

Takes place at a specific temperature of 70-74°C for about 5-15 minutes immediately after the last PCR cycle tasked in extending any remnants single DNA as its primary objective.

6. Final hold.

The overall mixture cools to a temperature of 4-15°C, summing up the completion of the reaction, which is later followed by the short-term storage of the reaction.

Figure 3: Stages of Polymerase Chain Reaction

The three main stages of PCR further explain the adequate understanding of the PCR principles and procedure;

1. Exponential amplification.

Each cycle’s results constitute a double numeric of the desired segment present at the end of the previous cycle showing that the number of DNA to be obtained depends on the PCR cycles, affirming PCR is an exponential process.

2. Levelling off stage.

At this stage, the reaction tends to decelerate as the DNA polymerase loses activity and depletes common reagents such as primers and dNTPs.

3. Plateau stage.

This stage best describes the late PCR cycles and how the exponential rate of products decreases due to the stability and utilization of reactants and substrates such as dNTPs, enzymes, or primers.

Applications of Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is often associated with the successful scientific advances in molecular biology because of its fastness and inexpensiveness, hence continuing to be widely applied in vast areas of medical and biochemical research laboratories. Billions of DNA copies realized from PCR offer a wide range of uses, especially in forensic, identification, and detection of genetic diseases and comprehensive molecular genetics coverage. Some of the vital applications of PCR in the field of genetic research, medicine, forensic science, and environmental microbiology are discussed below;

Genetic Research

Scientists continue to carry relevant research in this field, leading to many specialized applications in genetics. Gene expression patterns represent one of the typical applications of PCR, allowing scientists to be able to understand the genetic make-up of a specific gene through the cells or tissue analysis).  The standard variant of qPCR has proved to be more effective in this area as it helps to ascertain the proportion of gene expression. Rising modern techniques such as DNA sequencing continue to be applied in genetic mutations that are evident in many genetic diseases such as phenylketonuria and sickle cell anaemia that causes detrimental effects.

The development of the Human Genome Project represents another application in this field. The project has made it possible for scientists to identify specific genome segments in an interested clone, thus enabling the mapping of experimental clones and assembling results from other laboratories. Pregnant women continue to benefit from the existing types of PCR as doctors can study and analyze the chromosomal structures that prove to be useful in the early detection of genetic birth abnormalities in children.

Medicine

Existing and vast substantial progress of PCR continues to gather pace in many medical disciplines such as microbiology, virology, mycology, parasitology, and dentistry. Microbiology benefits a lot from PCR, especially in genotyping development, as it helps in an organism’s awareness. A recent study of various organisms like Mycobacterium tuberculosis with genotyping provides cutting-edge findings and treatment, hence administering medicine.  General understanding of the virus’s behaviour during infection is another area in virology that PCR has continues to flourish.

PCR has, over the years, been used in detecting the Human Immunodeficiency Virus (a complicated virus) at an early stage, even before the formation of antibodies.  The knowledge also continues to thrive in screening blood samples stored during the donations processes. Mycology and parasitology have much enjoyed the recent PCR application, especially in proper diagnosis and treatment of parasitic and fungal contaminations. Periodontal diseases, oral cancer, and caries conditions have been medically managed accordingly with PRC and recent molecular biology developments in dentistry.

Forensic Science

High crime rates bring detrimental results to the human population. The emergence of PCR through DNA fingerprinting makes it possible for the microscopic fragments of DNA from a crime scene to be recovered hence playing a crucial role in convicting criminals as well as cancelling out suspects in an investigation.  Paternity testing is also another area in which PCR is applied in determining the biological parenthood of individuals under study. More importantly, recognition of an unidentified body has been made possible through genetic fingerprinting.

Environmental Microbiology

Many environmental microbiology issues, such as enhancing water safety by applying a gene probe-based PCR, help detect hazardous bacteria such as coliforms. The same concept is often used to identify and control water-borne microbial pathogens, ensuring the environment’s safe advancement. PCR is also applied in maintaining effective biodegradation and bioremediation by getting rid of toxic waste pollutants.

Machines Used for Polymerase Chain Reaction (PCR) Brands in the Market

PCR machines continue to develop with time making them compulsory to have in any scientific research laboratory hence creating an increment in demand in the global market. Depending on the research and financial capability objectives, an individual can choose from the variations of PCR models brands in the market. Common interest often lies between choosing an endpoint PCR, qrtPCR, or a more digital PCR device, but with the current improvements in the machines, and optimally configured rtPCR has proved in meeting most of the needs of the laboratories. Below represents the common types of PCR machines brands in the market and their features;

Thermo Fisher

Thermo Fisher QuantStudio 3 Real-Time PCR Systems, Thermo Fisher StepOne, and StepOnePlus Real-Time PCR System are some of the PCR brands’ models in the market. Thermo Fisher Scientific is regarded as one of the most reliable PCR systems that come with different features and price ranges in the market. Essential traits of being economical, relevant for unskilled users, high multiplexing capabilities, laboratory robotics, and the modern linkage with a mobile app continue to benefit scientists as they updated with the latest models consistently.

Bio-Rad Systems

This PCR machine comes with its ultimate unique features, making it a familiar brand for users. Its software allows it to be installed to more than one computer hence the possibility of using multiple user accounts in operating the system (Baker, 2012).  It also supports other typical applications such as Excel and PowerPoint, especially in plate setup and writing of reports, respectively. Geometric normalization in the multiplication of reference genes has also been made possible. Bio-Rad CFX96, CFX384, and CFX Connect Bio-Rad MyIQ and IQ5 represent existing brands in the market.

Roche

Roche PCR machines come with variations, and some of the common brands in the market are; Roche 480 Lightcycler, Roche Lightcycler 1536, Roche Lightcycler 2.0, Roche Mx3000p, and Mx3005p. Although they are familiar, these machines contain a modest throughput capacity that only functions with a 96-well block and requires an internal reference dye to reduce multiplexing options.

Eppendorf Mastercycler

Continue to develop a new series of brands in different generations. Mastercycler X50, Mastercycler Nexus, and Mastercycler Gradient brands are very flexible and allow up to 12 different temperatures across the block (Gerke, & Eppendorf, 2013). Low power consumption, touchscreen interface, flexible lid are among the features that are common to all the model brands of Eppendorf Mastercycler.

Takara TP800/TP860

These two brands complement one another with TP860 representing the modern brand in the market. Takara rtPCR is often prevalent in Asia and comes with a durable 96-well capacity that reads FAM and Texas red.

Qiagen Rotor-Gene 3000 and 6000

These PCR machines are among the most affordable and suitable for most genomic applications. Recently a Rotor-gene Q was introduced in the market and bragged as the only system with the capability of deciphering the most complicated class IV SNPs.  Rotorgene cyclers continue to face the common shortcoming of low throughout and the constant air-conditioning supply that makes it challenging to maintain under normal conditions.

Other examples of PCR machines include; Biometra T1 Thermocycler and Fluidigm Biomark. From the types of PCR discussed above offered by Biotech companies, scientists can best choose a relevant machine necessary for their projects. For instance, RotorGene 3 is best suited for low throughput and comes with an affordable budget, but others such as Thermo Fisher 7900HT proves to be very costly.

Scientific Breakthroughs of PCR as Core Lab Procedure

PCR, over the years, has transformed various medical and other fields associated with molecular biology through extraordinary discoveries and breakthroughs. The current Covid-19 pandemic draws much attention globally and remains to be seen if PCR can develop a vaccine that fights the virus. Scientists remain optimistic in making a breakthrough and continue rolling out the PCR test for SARS-CoV-2 to study the various mutations stages of the virus.

PCR’s more common success as a core lab procedure lies in drug developments for dangerous diseases such as diabetes, liver complications, Ebola, and Otitis media, among others. Through real-time RT-PCR new potential drugs have been evaluated and studied in the laboratory and compared with the cells to see how they respond. Known diabetes drugs that were discontinued in the market due to their high liver toxicity include troglitazone (Tro); previously sold as Rezulin, can easily be distinguished through RT2 Profiler PCR Arrays.

Real-time RT-PCR also played a vital role in utilizing fluorogenic probes meant to detect Ebola, showing another breakthrough of PCR. Another breakthrough lies in the new modern genetic testing field. Its ability to clearly distinguish microscopic variations in the DNA composition of different individuals allows the process to diagnose various mutations that could be inherited for future generations.

Mutant gene awareness and its probability of causing other related diseases also present another achievement of Polymerase Chain Reaction in the laboratory research.  PCR represents more accurate and reliable tests than standard tests, showing its breakthrough in detecting bacterial DNA in the offspring’s ear fluid, where culture methods had difficulty and complications in detecting it.  The ability of PCR to understand the organism’s DNA facilitates its recent breakthrough in favouring patients suffering from complicated diseases to have speedy treatment preventing further complications.

Conclusion

The above discussion on Polymerase Chain Reaction shows a highly promising future in the directions and its applications in various fields, more specifically in molecular biology. More awareness and inclusivity of all stakeholders tasked with laboratory testing and research should find a common platform in achieving a balance in phylogenetic and genetic diversity. Scientists should invest more modern Polymerase Chain Reaction machines that are more affordable and environmentally friendly as this will stimulate more breakthroughs in medical fields, assisting in combating diseases that are considered incurable. Additionally, new and current methods specialized in molecular biology should be implemented and more focused on identifying and detecting microorganisms and the diagnosis’s effectiveness and accuracy.