Exploring the Pros and Cons of Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a technique extensively employed in medicine and molecular biology research to generate numerous copies of a specific section of DNA, such as a particular gene. It serves various purposes, including initial DNA processing for sequencing and the generation of forensic DNA profiles from minuscule amounts of DNA. PCR also facilitates the identification of pathogens during infections by detecting the presence or absence of specific genes. For instance, during the COVID-19 pandemic, PCR tests determined whether a person's swab sample contained a segment of the virus's genetic material, providing a positive or negative infection result.

Developed by American biochemist Kary Mullis in 1983 while working for Cetus Corporation, PCR is a method routinely utilized to rapidly amplify a particular DNA sample, enabling scientists to increase the amount of DNA for comprehensive study. This technique plays a crucial role in a wide range of genetic testing and research methods, including the analysis of ancient DNA samples and the detection of infectious organisms. PCR involves a series of temperature-changing cycles that exponentially amplify even minute amounts of DNA sequences. Its applications span various fields, such as biological research and criminal forensics, making PCR a frequently employed tool in medical laboratory research.

PCR is indispensable for genetic testing and analysis, including the identification of infectious organisms and the estimation of DNA sample age. By undergoing multiple cycles of elemental changes, PCR enables the exponential amplification of extremely small DNA sequences. Its utilization in medical laboratory examinations has become increasingly commonplace and essential for purposes like biomedical research and criminal forensics.
Thermal cycling, a crucial component of most PCR techniques, involves temperature-dependent processes like DNA melting and enzyme-driven DNA replication. These processes ensure the efficient amplification of DNA sequences while protecting reactants during thermal cycling.

Taq polymerase, an enzyme derived from the thermophilic bacteria Thermus aquaticus, is utilized in almost all PCR applications. Taq polymerase is resistant to the high denaturation temperatures required during PCR, preventing its denaturation. Before the use of Taq polymerase, DNA polymerase had to be manually added to each cycle, a time-consuming and costly process.

PCR Components

PCR consists of several key components:

• DNA template: The starting material, which is the DNA from the sample of interest.
• DNA polymerase: Taq Polymerase is commonly used because it is heat-resistant and remains active at high temperatures without denaturing.
• Primer oligonucleotides: These short, single-stranded DNA sequences with complementary sequences to the target DNA's sense and antisense strands serve as starting points for DNA synthesis.

•Deoxyribonucleotide Triphosphates (dNTPs): These are the building blocks for DNA synthesis and provide stability during polymerization. They are unpaired bases.
• Buffer system: The presence of magnesium and potassium in the buffer enhances DNA denaturation and renaturation, and improves polymerase activity and stability.

Types of PCR:

PCR can be performed in different formats:

• Real-time PCR: This technique uses a fluorescent probe to monitor DNA amplification in real time. The strength of the fluorescent signal from the probe directly correlates with the amount of amplified DNA molecules present.
• Nested PCR: This method aims to increase specificity and sensitivity by using additional sets of primers.By targeting different regions, it reduces the non-specific binding of unwanted DNA fragments.
• Multi-step PCR: With this approach, multiple target DNA sequences can be amplified in a single PCR experiment. It allows for the simultaneous amplification of different DNA targets.
• Quantitative PCR: This method utilizes the linear amplification of DNA to identify, characterize, and quantify a specific target within a sample.

Advantages of PCR:

PCR offers several advantages. It is user-friendly and easily understandable, and it operates with speed. It enables the production of a specific product in millions to billions of copies using a highly sensitive approach for sequencing, cloning, and analysis. QRT-PCR shares the advantages of PCR but additionally provides the ability to quantify the results. As a result, it finds applications in studying changes in gene expression levels in tumors, bacteria, and other disease states.

PCR serves as an immensely powerful and valuable research tool. It is employed to determine the sequence of diseases with unknown etiologies, shedding light on the diseases themselves and uncovering sequences of previously unidentified viruses linked to known ones. If the process can be further simplified and sensitive non-radiometric detection techniques can be developed, PCR will continue to hold a crucial position in clinical laboratories for years to come.
• PCR presents challenges in calculating the regions of DNA throughout the denaturation, amplification, and replication cycles.
• The use of specific DNA primers suggests the identity of the targeted bacterium precisely. In the field of molecular biology, PCR has emerged as a dominant technique, facilitating gene identification and mutation detection, thereby enhancing researchers' capabilities. 
• It has evolved into a method for identifying microbial pathogens with minimal sample representation required for investigation. Currently, PCR is primarily utilized for confirmatory investigations in the diagnosis of herpetic keratitis. 
• The PCR procedure takes approximately 4-8 hours, which is roughly three hours shorter than conventional methods. 
• Multiplex PCR, a specific type of PCR, allows for the amplification of multiple DNA targets simultaneously. It has gained recognition as the gold standard for the diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) due to its ability to detect various antibiotic resistance genes produced by MRSA.

Disadvantages of PCR:

PCR has several significant limitations. One major drawback is the need for prior knowledge of the target sequence in order to design specific primers for selective amplification. PCR users must be aware of the precise sequences upstream of the target region on both single-stranded templates to ensure accurate binding of the DNA polymerase to the primer-template hybrids and successful amplification of the entire target region during DNA synthesis.

Like other enzymes, DNA polymerases can introduce errors, leading to mutations in the PCR fragments produced. Another disadvantage of PCR is the possibility of false-positive or inconclusive results, even with minute amounts of contaminated DNA. To minimize the risk of contamination, reagent preparation, PCR setup, and result analysis should be conducted in separate rooms. Single-use aliquots of reagents should be used, and pipettors with extra-long pipette tips and disposable plungers are recommended. It is also important to follow a unidirectional workflow in the laboratory and thoroughly decontaminate any supplies or reagents before entering the PCR preparation room from other rooms used for PCR or result analysis.

• PCR has certain limitations, including lower specificity compared to culturing and staining, which increases the likelihood of false positives. Complex primer design and the need to consider multiple potential microbes before performing intricate procedures have been challenges for doctors. 
• The primers used for infectious keratitis, such as the 16S bacterial DNA primers and the 18S fungal DNA primers, are well-known. However, due to similarities between different bacteria and fungi, there is a risk of amplifying shared DNA, leading to false positives when detecting normal flora from the external environment of the cornea.
• In one instance, unrelated microbes were detected in control PCR samples, potentially due to airborne contamination during sample shipment from India. Although airborne contamination is rare in PCR, it may be more common in keratitis cases or considered an exceptional finding.
• Cross-contamination is a concern in both culturing and staining, but the sensitivity of PCR increases this risk. Different geographical regions may harbor distinct microbial populations.
 • In a ten-year investigation, it was reported that MRSA-related keratitis was more severe in a specific area compared to other provinces, suggesting that clinicians should consider regional factors when requesting and interpreting PCR results.
• DNA polymerases, although highly efficient, are not flawless. They can introduce errors both in the human body and during PCR. For example, Taq polymerase, commonly used in PCR, lacks 3' to 5' exonuclease activity, which prevents the correction of incorrectly incorporated nucleotides, a feature present in higher eukaryotes.

Challenges and Potential of PCR-Based Metabarcoding in Ecological Sampling

PCR technology plays a crucial role in metabarcoding investigations, but the outcomes differ significantly from conventional non-PCR-based sampling methods. This results in several important observations. First, there is no direct relationship or one-to-one correspondence between the number of assigned reads in an eDNA study and the abundance of the source organism. Second, we cannot consistently expect a high correlation in estimates of taxon-richness between eDNA and traditional methods. Even small adjustments in laboratory procedures can lead to large discrepancies in results.

Despite these challenges, metabarcoding surveys have proven to be highly useful. They enable the detection of hundreds or thousands of taxa in each sample and facilitate the identification of biological communities in different habitats and sampling locations. However, for eDNA to become a common source of data in ecological sampling, it is crucial to understand the mechanisms that link amplicon reads to species' biomass or counts. This understanding is necessary for applications such as fisheries stock assessments or population surveys for endangered species, which require quantification of organisms.

By focusing on the generation of metabarcoding data, we have gained insights into the specific ways in which these data can and cannot be compared to other survey approaches. In doing so, we have provided a quantifiable mechanism to monitor changes in environmental samples