1. Introduction

DOI: 10.52228/NBW-JAAB.2020-2-1-3

PCR is a widely used molecular biology technique for amplifying specific genomic DNA sequences. It was first developed by Kary Mullis in 1983 and has since become a cornerstone technique in molecular biology research and clinical diagnostics. PCR works by using a heat-stable DNA polymerase enzyme, which replicates a targeted DNA sequence through a series of temperature cycles. The process involves the denaturation of double-stranded DNA into single-stranded DNA, annealing of primers to the single-stranded DNA, and extension of the primers by the DNA polymerase to create a new strand of DNA. This process is repeated multiple times, leading to exponential amplification of the targeted DNA sequence (Saiki et al., 1985).

The applications of PCR are diverse and include gene expression analysis, genetic testing, forensics, and pathogen detection. PCR is also used in conjunction with other techniques such as gel electrophoresis, DNA sequencing, and restriction enzyme analysis. PCR has revolutionized the field of molecular biology by allowing the amplification of small amounts of DNA, which was previously not possible. This technique has enabled researchers to study genetic information in detail and has contributed to significant advances in fields such as genetics, genomics, and biotechnology (Orlando et al., 2015).

In addition to its applications in research, PCR has significant implications for clinical diagnostics. PCR-based diagnostic tests are used to detect pathogens, identify genetic mutations, and screen for inherited disorders. PCR is also used in forensic science to identify suspects and analyse DNA evidence. The purpose of PCR is to amplify a specific DNA sequence from a small amount of starting material. PCR enables the rapid and precise amplification of a targeted DNA sequence through a series of temperature cycles. The amplified DNA can be used for a variety of purposes, including genetic testing, gene expression analysis, forensics, pathogen detection, and more.  PCR can amplify DNA sequences ranging in size from a few hundred base pairs to several kilobases. By using specific primers that hybridize to the flanking regions of the target DNA sequence, PCR selectively amplifies only the desired DNA fragment, even in the presence of other DNA templates (Templeton et al., 2013). PCR has numerous applications in various fields, including research, medicine, and forensics. For example, PCR can be used to detect the presence of specific DNA sequences associated with disease, identify genetic mutations, or diagnose infectious diseases caused by bacteria or viruses. PCR can also be used to study gene expression patterns or create large quantities of a specific DNA fragment for downstream applications, such as cloning or sequencing. the purpose of PCR is to rapidly and precisely amplify a specific DNA sequence, which has enabled significant advances in molecular biology research and clinical diagnostics (Freire et al., 2012).  Since its inception, PCR has undergone several modifications and improvements, such as the introduction of quantitative PCR (qPCR), digital PCR (dPCR), and reverse transcription PCR (RT-PCR), to name a few.

1.1 Historical background

PCR was invented by Kary Mullis in 1983 and has since revolutionized the field of molecular biology. The development of PCR has had a profound impact on many areas of research, including genetics, genomics, biotechnology, and clinical diagnostics. Kary Mullis was working as a chemist at Cetus Corporation when he developed PCR. The idea for PCR came to him while he was driving on a highway and thinking about a way to copy DNA. Mullis realized that he could use temperature cycling to rapidly and accurately amplify a specific DNA sequence (Mullis, 1990).

The first publication describing PCR was in 1985 in the journal Science, where Mullis and his colleagues demonstrated the use of PCR to amplify the beta-globin gene. This publication marked the beginning of a new era in molecular biology, where small amounts of DNA could be amplified to obtain enough material for analysis.  Since its development, PCR has undergone numerous modifications and improvements, including the use of thermostable DNA polymerases, fluorescent probes, and digital PCR. These advances have expanded the range of applications of PCR and made it an indispensable tool in molecular biology research and clinical diagnostics (Matsuda et al., 2017).

The development of PCR occurred during a time of rapid advances in molecular biology, genetics, and biotechnology. The groundwork for PCR was laid in the 1960s and 1970s, with the development of recombinant DNA technology, which allowed for the manipulation of DNA sequences in vitro. In the early 1980s, DNA sequencing techniques had become available, allowing researchers to determine the sequence of DNA fragments.  The idea for PCR came to Kary Mullis while he was working at Cetus Corporation, a biotechnology company in California, in the early 1980s. Mullis was trying to find a way to copy DNA without using bacteria, which was the traditional method for amplifying DNA at the time. His solution was to use a thermostable DNA polymerase and a series of temperature cycles to amplify specific DNA sequences (Mullis, 1994).

The development of PCR was a significant breakthrough in molecular biology, as it allowed researchers to amplify DNA sequences in a matter of hours, rather than days or weeks. The first publication describing PCR was in 1985 in the journal Science, where Mullis and his colleagues demonstrated the use of PCR to amplify the beta-globin gene. This publication marked the beginning of a new era in molecular biology, where small amounts of DNA could be amplified to obtain enough material for analysis.  The impact of PCR has been so profound that Kary Mullis was awarded the Nobel Prize in Chemistry in 1993 for his invention. Today, PCR has continued to evolve with advances such as real-time PCR, digital PCR, and high-throughput PCR, expanding its range of applications and increasing its sensitivity and accuracy (Bartlett et al., 2003).

1.2 Experiments that led to its Development and the key players involved

One of the key experiments that led to the development of PCR was performed by Saiki et al. in 1985, where they demonstrated that PCR could be used to amplify a specific DNA sequence from humans' DNA. They used primers to select the target DNA sequence and a heat-stable DNA polymerase to enable amplification through cycles of heating and cooling. The success of this experiment provided proof of the principle that PCR could be used to amplify specific DNA sequences. Another key player in the development of PCR was Henry Erlich, a researcher at Cetus Corporation, who worked with Mullis to optimize the reaction conditions for PCR (Kim & Smithies, 1985). Erlich played a critical role in developing the technology for amplifying DNA from small amounts of starting material, which was a major limitation of earlier methods for DNA amplification.  The discovery of thermostable DNA polymerases, such as Taq polymerase, was also a critical development in the early years of PCR. These enzymes, isolated from bacteria that live in hot springs, were able to withstand the high temperatures used in PCR cycles, making the process more robust and efficient (Ishino & Ishino, 2014).

1.3 Evolution of PCR over time and the various modifications

The development of PCR was a collaborative effort involving many researchers, including Kary Mullis, Henry Erlich, and Saiki et al. Their contributions laid the foundation for the widespread use of PCR in molecular biology research and clinical diagnostics. PCR has undergone significant evolution and modification since its invention in the early 1980s, leading to a variety of new techniques and applications. Some of the major advancements in PCR technology are discussed below, along with relevant references (Sachefe et al., 2006).

Real-time PCR: In the mid-1990s, real-time PCR was developed, which allowed for the detection and quantification of PCR products in real-time. This technology utilizes fluorescent dyes or probes to detect the amplification of DNA in real-time, enabling researchers to monitor the progress of PCR in real-time, without the need for post-PCR analysis. Real-time PCR is widely used in quantitative PCR (qPCR) and gene expression analysis (Higuchi et al., 1993) followings

i.     Nested PCR: Nested PCR is a modification of PCR that involves two sets of primers. The first set amplifies a larger DNA fragment, while the second set amplifies a smaller fragment within the first PCR product. Nested PCR is used to increase specificity and sensitivity by amplifying a specific target from a complex mixture, and it has applications in medical diagnosis, forensics, and microbiology (Llop et al., 2000).

ii.     Reverse transcription PCR (RT-PCR): RT-PCR is a modification of PCR that is used to amplify RNA sequences. This technique involves the reverse transcription of RNA into complementary DNA (cDNA), followed by PCR amplification. RT-PCR is commonly used to study gene expression and viral infections (Overbergh et al., 2003).

iii.     Multiplex PCR: Multiplex PCR is a modification of PCR that enables the amplification of multiple targets in a single reaction. This technique utilises multiple primer sets, each specific to a different target DNA sequence, and is commonly used in the diagnosis of genetic disorders and infectious diseases.

iv.     Digital PCR: Digital PCR is a recent advancement in PCR technology that allows for the absolute quantification of nucleic acid targets without the use of standard curves. This technique involves the partitioning of a sample into thousands of tiny reactions, each containing one or no DNA molecules. The presence or absence of the target DNA sequence is detected by end-point PCR analysis, and the total number of target molecules is calculated using Poisson statistics.

v.     Loop-mediated isothermal amplification (LAMP): LAMP is an alternative to PCR that amplifies DNA under isothermal conditions, meaning that a constant temperature is used throughout the amplification process. LAMP utilises multiple primers to amplify DNA in a loop-mediated manner, resulting in high sensitivity and specificity. This technique is used for the diagnosis of infectious diseases and genetic disorders (Khamlor et al., 2015).

1.4 PCR Basic

Overall, the evolution of PCR has led to a wide range of modifications and advancements that have greatly expanded its applications and usefulness in molecular biology and other fields. PCR is a widely used molecular biology technique that allows for the amplification of specific DNA sequences. The basic principles of PCR involve three steps: denaturation, annealing, and extension. These steps are repeated multiple times to exponentially amplify the target DNA sequence. Below is a detailed description of the basic principles of PCR, along with relevant references (Joshi & Deshpande, 2010),

1.4.1 Denaturation

Denaturation is the first step of the PCR, a widely used technique in molecular biology that allows for the amplification of specific DNA sequences. In denaturation, the double-stranded DNA (dsDNA) template is heated to a high temperature, usually around 94-98°C, causing the hydrogen bonds between the two complementary strands of DNA to break. This results in two single-stranded DNA (ssDNA) templates. During denaturation, the high temperature disrupts the hydrogen bonds between the two strands of the dsDNA template, causing the two strands to separate and form two individual ssDNA templates. The temperature and duration of the denaturation step are critical for successful amplification, as they affect the efficiency and specificity of the PCR.

The denaturation step in PCR typically lasts for 15-30 seconds, depending on the length and complexity of the DNA template. If the temperature is not high enough or the denaturation time is too short, the dsDNA template may not fully denature, leading to incomplete separation of the two strands and reduced efficiency of the subsequent steps in PCR (Lorenz 2012). On the other hand, if the temperature is too high or the denaturation time is too long, the DNA template may become damaged or degraded, leading to reduced specificity and increased background noise.  Denaturation is a critical step in PCR, as it allows the primers to bind specifically to their complementary sequences on the ssDNA templates in the subsequent step of PCR. The specificity of PCR depends on the primers' ability to bind specifically to the target DNA sequence. By separating the two strands of the dsDNA template, the primers can bind to their complementary sequences on the ssDNA templates with high specificity, allowing for the selective amplification of the target DNA sequence. Denaturation is a critical step in the PCR process that separates the two strands of the dsDNA template, allowing the primers to bind specifically to their complementary sequences on the ssDNA templates. The temperature and duration of the denaturation step must be carefully controlled to ensure successful and specific amplification of the target DNA sequence (Saiki et al., 1988).

1.4.2 Annealing

This is the second step in the PCR, a technique used to amplify specific regions of DNA. During annealing, the temperature of the reaction mixture is lowered to allow the primers, short DNA sequences that bind to specific regions of the target DNA, to anneal to the complementary sequences on the single-stranded DNA templates generated during the denaturation step (Green et al., 2015). The annealing temperature used in PCR is critical for efficient and specific binding of the primers to their complementary sequences on the template DNA. The annealing temperature is typically a few degrees Celsius below the melting temperature (Tm) of the primers, which is the temperature at which 50% of the primer is bound to its complementary sequence on the template DNA. The Tm of the primers is influenced by the length, sequence, and GC content of the primers. A lower annealing temperature increases the chances of nonspecific binding, while a higher annealing temperature may reduce the efficiency of annealing (Liu et al., 2008).

The annealing step is typically done for a short period of time, typically 15-30 seconds. During this time, the primers bind to their complementary sequences on the template DNA through hydrogen bonding, forming a stable primer-template complex. The specificity of PCR is largely determined by the annealing step, as it ensures that the primers only bind to their complementary sequences on the template DNA, preventing the amplification of unintended sequences. Once the primers have annealed to the template DNA, the reaction temperature is increased to the extension temperature, which allows the polymerase enzyme to synthesize new DNA strands using the primers as templates. The annealing step is repeated in each cycle of the PCR process, allowing the primers to bind to newly generated single-stranded template DNA after each denaturation step.

In summary, the annealing step in PCR is a critical component of the PCR process, allowing primers to bind specifically to their complementary sequences on the template DNA. The annealing temperature and duration must be carefully Optimised to ensure efficient and specific binding of the primers to their target sequences, enabling amplification of the desired DNA region (Rand et al., 2005).

1.4.3 Extension

The extension is the third step in the PCR, a technique used to amplify specific regions of DNA. During extension, the temperature of the reaction mixture is raised to allow the polymerase enzyme to synthesize new DNA strands using the primers as templates. The polymerase used in PCR is typically a thermostable DNA polymerase, such as Taq polymerase, which is derived from the bacterium Thermus Aquatics. Taq polymerase is able to withstand the high temperatures used in PCR, making it ideal for the extension step. Other thermostable polymerases, such as Pfu and Phusion, may also be used in PCR and offer higher fidelity than Taq polymerase. The extension temperature used in PCR is typically between 68-72°C, which is optimal for Taq polymerase activity. During the extension step, the Taq polymerase synthesizes new DNA strands by adding nucleotides to the 3’ end of the primers. The extension time required for the complete synthesis of the new DNA strands depends on the length of the target DNA fragment and the activity of the polymerase enzyme used. Typically, extension times range from 30 seconds to 2 minutes per kilobase of DNA.

The synthesis of new DNA strands during the extension step is a critical component of the PCR process, as it allows for exponential amplification of the target DNA fragment. Each cycle of PCR results in the generation of new copies of the target DNA fragment, with the number of copies increasing exponentially with each cycle (Mullis et al., 1992). Once the extension step is complete, the temperature is lowered to begin the next cycle of PCR, which involves denaturation of the newly synthesized double-stranded DNA molecules, followed by annealing and extension of the primers to generate new DNA copies.

In summary, the extension step in PCR is a critical component of the PCR process, allowing the polymerase enzyme to synthesize new DNA strands using the primers as templates. The extension temperature and time must be carefully optimised to ensure efficient and specific amplification of the target DNA fragment. After the first cycle of denaturation, annealing, and extension, the resulting DNA strands become templates for the subsequent cycles, resulting in exponential amplification of the target DNA sequence. The number of cycles can be adjusted to achieve the desired level of amplification. PCR has become a powerful tool in molecular biology, with applications ranging from medical diagnosis to genetic research. The basic principles of PCR have remained the same since its invention, but the technique has undergone significant modifications and improvements over time, leading to the development of new variants such as real-time PCR and digital PCR (Mao et al., 2019).

1.4.4 PCR Applications

The following applications of PCR are discussed in this paragraph

1.4.4.1 Diagnostics

PCR has become an important tool in the field of diagnostics due to its high sensitivity and specificity. In diagnostic applications, PCR is used to detect the presence of nucleic acids from pathogens or genetic mutations associated with diseases in patient samples such as blood, urine, and tissue samples. The amplification of DNA/RNA sequences by PCR can be used to detect even a small amount of pathogenic or mutant DNA/RNA in a sample, making it a highly sensitive diagnostic tool.

One of the most common diagnostic applications of PCR is in infectious disease testing. PCR can be used to detect the presence of viral, bacterial, and fungal pathogens in clinical samples. For example, PCR is commonly used for the diagnosis of sexually transmitted infections (STIs) such as chlamydia, gonorrhoea, and human papillomavirus (HPV). PCR can also be used for the detection of respiratory viruses such as influenza, coronavirus, and respiratory syncytial virus (RSV). Another application of PCR in diagnostics is in the detection of genetic mutations associated with inherited diseases such as cystic fibrosis and sickle cell anaemia. PCR can be used to amplify specific regions of the patient's DNA to identify mutations associated with the disease. Real-time PCR, also known as quantitative PCR (qPCR), is another diagnostic application of PCR that allows for the quantification of nucleic acid sequences in a sample. Real-time PCR can be used to monitor the progression of infections, track disease outbreaks, and monitor treatment efficacy.  PCR-based assays have also been developed for non-infectious diseases such as cancer. These assays can be used to detect genetic mutations associated with cancer in patient samples, such as the detection of the BRAF V600E mutation in melanoma or the EGFR mutation in lung cancer. Overall, PCR has become an essential tool in the diagnosis of infectious and genetic diseases, as well as in cancer diagnosis and monitoring. Its high sensitivity, specificity, and ability to detect a small amount of nucleic acid make it a powerful diagnostic tool in the field of medicine (Kaltenboeck & Wang, 2005).

1.4.4.2 Genetic testing

PCR has become a powerful tool in genetic testing due to its ability to amplify specific DNA sequences of interest from small samples. Genetic testing using PCR can be used to identify genetic mutations associated with inherited diseases or to identify genetic variations that predispose individuals to certain diseases or conditions. One of the most common types of genetic testing using PCR is the detection of mutations associated with inherited diseases. PCR can be used to amplify specific regions of DNA that are associated with the disease, allowing for the identification of the mutation. For example, PCR can be used to identify the F508del mutation in the CFTR gene associated with cystic fibrosis.  PCR-based genetic testing can also be used to identify genetic variations that increase an individual's risk of developing certain diseases. For example, PCR can be used to detect the presence of the BRCA1 and BRCA2 mutations associated with an increased risk for breast and ovarian cancer. Real-time PCR, also known as quantitative PCR (qPCR), has been used in genetic testing to quantify the expression levels of genes associated with certain diseases. For example, qPCR can be used to measure the expression levels of genes associated with cancer, allowing for the identification of genes that are overexpressed or under-expressed in cancer cells. In addition to identifying genetic mutations associated with diseases, PCR can also be used for paternity testing and forensic testing. PCR-based testing of short tandem repeat (STR) sequences can be used to determine paternity or to match DNA evidence to a suspect in a criminal investigation. PCR has revolutionized genetic testing by allowing for the rapid and accurate identification of genetic mutations associated with inherited diseases or an increased risk of developing certain diseases. Its high sensitivity and specificity make it an essential tool in the field of genetics (Neff et al., 1998).

1.4.4.3 Forensic analysis

PCR has become a powerful tool in forensic analysis due to its ability to amplify specific DNA sequences from small samples, allowing for the identification of individuals based on their DNA profiles. PCR-based forensic analysis can be used in a variety of applications, including DNA profiling, paternity testing, and identification of remains. One of the most common applications of PCR in forensic analysis is DNA profiling, which is also known as DNA fingerprinting. PCR can be used to amplify short tandem repeat (STR) sequences from a small amount of DNA found at a crime scene, allowing for the creation of a DNA profile that is unique to each individual. The DNA profile can then be compared to DNA samples collected from suspects to determine if they were present at the crime scene.  PCR can also be used in paternity testing to determine the biological relationship between individuals. PCR-based testing of STR sequences can be used to determine the probability of paternity based on the presence or absence of specific genetic markers.

Another application of PCR in forensic analysis is the identification of human remains. PCR can be used to amplify mitochondrial DNA (mtDNA) sequences from small amounts of bone or tissue, allowing for the identification of individuals even when traditional DNA profiling is not possible.  Real-time PCR, also known as quantitative PCR (qPCR), has also been used in forensic analysis to quantify the expression levels of genes associated with certain diseases or conditions. For example, qPCR can be used to measure the expression levels of genes associated with aging, allowing for the determination of an individual's chronological age based on their gene expression profile.

PCR has revolutionized forensic analysis by allowing for the rapid and accurate identification of individuals based on their DNA profiles. Its high sensitivity and specificity make it an essential tool in the field of forensics.  Gene expression analysis: PCR can be used to analyze gene expression by measuring the amount of mRNA produced by a gene of interest. This technique, known as reverse transcription PCR (RT-PCR), involves the conversion of RNA to complementary DNA (cDNA) using the enzyme reverse transcriptase. The resulting cDNA can then be amplified using PCR with primers specific to the gene of interest (Kashyap et al., 2004).

1.4.4.4 Gene expression:

RT-PCR can be used to analyse gene expression in a variety of applications, including disease diagnosis and drug development. For example, RT-PCR can be used to measure the expression levels of genes associated with cancer, allowing for the identification of potential targets for therapy.  Real-time PCR, also known as quantitative PCR (qPCR), is a variation of PCR that allows for the quantification of gene expression in real-time. In qPCR, fluorescent dyes are used to monitor the amount of PCR product produced during amplification. The fluorescence signal is proportional to the amount of PCR product, allowing for the precise measurement of gene expression levels (Lianidou, 2016).

qPCR can be used to analyse gene expression in a variety of samples, including blood, tissues, and cell cultures. It is a sensitive and accurate method for quantifying gene expression and can be used in both research and clinical settings.  In addition to traditional PCR and qPCR, there are several other variations of PCR that can be used for gene expression analysis, including nested PCR, multiplex PCR, and digital PCR. Each of these techniques has its own advantages and disadvantages, and the choice of technique will depend on the specific application and experimental design. PCR-based gene expression analysis has become an essential tool in the study of gene function and regulation, allowing for the identification of potential therapeutic targets (Jain et al., 2006).

1.4.4.5 Environmental monitoring

PCR-based techniques have become widely used in environmental monitoring to detect and quantify the presence of microorganisms and pollutants in environmental samples. Some of the most common PCR-based techniques used in environmental monitoring include-

●     Quantitative PCR (qPCR): qPCR is a real-time PCR-based technique that can be used to quantify the abundance of specific microorganisms in environmental samples. This technique is particularly useful in monitoring the abundance of pathogenic microorganisms in water and soil samples.

●     Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE): PCR-DGGE is a PCR-based technique that separates DNA fragments based on their melting behaviour during denaturation. This technique can be used to profile the microbial communities present in environmental samples and track changes in microbial diversity over time (Diallo et al., 2004).

●     Next-generation sequencing (NGS): NGS-based techniques, such as metagenomics and meta transcriptomics, can be used to identify and quantify the diversity of microorganisms present in environmental samples. These techniques can provide a comprehensive view of the microbial community and allow for the identification of novel microorganisms and potential pollutants.

●     Loop-mediated isothermal amplification (LAMP): LAMP is a PCR-based technique that can amplify DNA at a constant temperature, making it useful for field-based environmental monitoring. LAMP can be used to detect a wide range of microorganisms, including bacteria, viruses, and fungi. PCR-based techniques have been used in a variety of environmental monitoring applications, including water quality monitoring, soil quality monitoring, and air quality monitoring. For example, qPCR has been used to detect the presence of pathogenic bacteria, such as Escherichia coli, in water samples. PCR-DGGE has been used to track changes in microbial community composition in response to environmental disturbances, such as oil spills. NGS-based techniques have been used to identify novel microorganisms and potential pollutants in environmental samples. PCR-based techniques have revolutionized environmental monitoring, providing sensitive and accurate methods for detecting and quantifying microorganisms and pollutants in environmental samples (Parida et al., 2008).

●     Cancer research: PCR-based techniques have become essential tools in cancer research, allowing for the detection and analysis of cancer-related genetic alterations. Some of the most common PCR-based techniques used in cancer research include-

o Mutation detection: PCR can be used to detect mutations in cancer-related genes, such as TP53, BRAF, and KRAS. PCR-based techniques can be used to detect mutations in a variety of sample types, including tissue biopsies, blood samples, and circulating tumor cells.

o Gene expression analysis: PCR-based techniques can be used to measure the expression levels of cancer-related genes, allowing for the identification of genes that are upregulated or downregulated in cancer cells compared to normal cells. Real-time PCR-based techniques, such as qPCR, can be used to quantify gene expression levels with high sensitivity and accuracy.

o Microsatellite instability (MSI) analysis: MSI is a hallmark of some types of cancer, including colorectal cancer. PCR-based techniques can be used to detect MSI by amplifying short tandem repeat (STR) sequences that are prone to a mutation in MSI-positive tumours.

o Epigenetic analysis: PCR-based techniques can be used to analyse epigenetic modifications, such as DNA methylation and histone modifications that play important roles in cancer development and progression. Methylation-specific PCR (MSP) is a PCR-based technique that can be used to detect DNA methylation at specific CpG sites in cancer-related genes (Cheung et al., 2009). PCR-based techniques have been used in a variety of cancer research applications, including cancer diagnosis, prognosis, and treatment. For example, PCR-based mutation detection has been used to guide targeted therapies for patients with certain types of cancer, such as melanoma and lung cancer. Gene expression analysis using PCR-based techniques has been used to identify novel biomarkers for cancer diagnosis and prognosis. MSI analysis using PCR-based techniques has been used to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC). Epigenetic analysis using PCR-based techniques has been used to identify potential targets for epigenetic therapies in cancer. PCR-based techniques have played a critical role in cancer research, providing sensitive and accurate methods for detecting and analysing cancer-related genetic alterations (Soda et al., 2012).

1.4.4.6 Evolutionary biology

PCR has found numerous applications in the field of evolutionary biology, particularly in the study of genetic diversity, population genetics, and phylogenetics. The ability to amplify specific DNA sequences from diverse organisms has greatly facilitated the study of evolutionary relationships and the analysis of genetic variation within and between populations (Tylor et al., 1999). One of the major applications of PCR in evolutionary biology is the analysis of DNA sequence variation to infer evolutionary relationships and construct phylogenetic trees. This involves the amplification of specific DNA sequences from different taxa using PCR, followed by sequencing and alignment of the sequences to identify similarities and differences. These data can then be used to reconstruct evolutionary relationships and infer ancestral states, providing insights into the origins and diversification of different groups of organisms.  PCR has also been used to study population genetics, by amplifying and analysing DNA sequences from different individuals within populations. This allows researchers to estimate genetic diversity and infer patterns of migration, gene flow, and selection within and between populations (Moritz, 2004).

1.4.4.7 Food safety

PCR has become an increasingly important tool in ensuring the safety of the food supply. The ability to rapidly and accurately detect the presence of harmful pathogens or contaminants in food products has greatly improved food safety and reduced the risk of foodborne illness outbreaks. One of the primary applications of PCR in food safety is the detection of foodborne pathogens, such as Salmonella, E. coli, and Listeria. PCR can be used to amplify and detect specific DNA sequences from these pathogens, allowing for rapid and accurate identification of their presence in food products (Kumar & Thakur, 2018). PCR has also been used to detect other types of contaminants in food, such as genetically modified organisms (GMOs) and allergens. By amplifying and detecting specific DNA sequences associated with these contaminants, PCR can help ensure that food products are properly labeled and meet regulatory standards.  In addition to these applications, PCR has been used to study the microbiome of food products, providing insights into the diverse community of microorganisms that inhabit different types of food. This can help identify potential sources of contamination and inform strategies for improving food safety. PCR has become an essential tool in ensuring the safety and quality of the food supply, allowing for rapid and accurate detection of harmful pathogens and contaminants (Chiang et al., 2014).

1.4.4.8 Genotyping in PCR

PCR is widely used in genetic research and clinical diagnosis for genotyping, or the identification of specific genetic variations in an individual's DNA. Genotyping using PCR can help researchers understand the genetic basis of diseases and develop personalized treatment strategies based on an individual's genetic makeup. One common application of PCR-based genotyping is in the detection of single nucleotide polymorphisms (SNPs), or variations in a single nucleotide at a specific location in the DNA sequence. PCR can be used to amplify a specific DNA fragment containing the SNP, and then sequence the fragment to determine whether the SNP is present or absent. This can be used for a range of applications, from studying genetic disease risk to predicting drug response.   Another application of PCR-based genotyping is in the detection of genetic mutations associated with specific diseases, such as cystic fibrosis or sickle cell anaemia. PCR can be used to amplify specific regions of the gene associated with the disease, and then sequence the fragment to identify mutations that may cause or contribute to the disease (Syvanen, 2001).

1.4.4.9 Infectious Disease Diagnosis in PCR

PCR has become an essential tool in the diagnosis of infectious diseases, providing rapid and accurate detection of pathogenic microorganisms in clinical samples. PCR-based diagnostic tests can be used for a wide range of infectious agents, including bacteria, viruses, and parasites. PCR-based diagnosis of infectious diseases typically involves amplifying and detecting specific DNA or RNA sequences associated with the pathogen in a patient sample. For example, in the case of a bacterial infection, PCR can be used to amplify and detect specific genes or genomic regions that are unique to the bacterial species or strain causing the infection.  PCR-based diagnostic tests have several advantages over traditional culture-based methods for detecting infectious diseases. They can provide faster results, as PCR can detect even small amounts of pathogen DNA or RNA in a sample, whereas culture-based methods may require several days to produce a positive result. PCR-based tests are also more sensitive and specific than traditional methods, allowing for more accurate diagnosis and treatment of infectious diseases (Natomi et al., 2000).

1.5    PCR Optimization

PCR optimization is the process of adjusting various parameters in a PCR reaction to achieve optimal results. Optimization is important to ensure that the reaction is specific, sensitive, and efficient, leading to accurate and reproducible results (Rowlands et al., 2019). Here are some factors that can be optimized in a PCR reaction:

Ø Primer design: Primer design is a critical step in PCR, as it determines the specificity, efficiency, and sensitivity of the reaction. Primers are short, single-stranded oligonucleotides that anneal to complementary sequences flanking the target region, providing a template for DNA polymerase to extend and synthesize the new complementary strand. Here, we will discuss the key principles and strategies for primer design in PCR.

Ø Target region selection: The first step in primer design is to identify the target region of interest. The target region should be unique and specific to the intended template, avoiding regions of homology with other sequences in the sample. The length of the target region should also be considered, as longer regions may require higher annealing temperatures and longer primers.

Ø Primer length and Tm: The length and melting temperature (Tm) of the primers are crucial for their binding specificity and efficiency. The recommended primer length is between 18-22 nucleotides, with a Tm of around 55-60°C. The Tm of the primers should be close to each other to ensure similar annealing efficiency. Various online tools and software are available to calculate the Tm of primers based on their sequence composition, such as OligoCalc and Primer-3.

Ø GC content and secondary structure: The GC content of the primers should be between 40-60%, as too high or too low GC content can affect their annealing efficiency and specificity. The presence of secondary structures such as hairpins or dimers can also hinder primer annealing and extension, leading to non-specific amplification or failure of the reaction. Online software such as IDT OligoAnalyzer and PrimerQuest can help predict and optimize primer secondary structures.

Ø Primer specificity: The primers should be designed to minimize the possibility of binding to non-target sequences, such as homologous genes or repetitive elements. Several online databases, such as BLAST and UCSC Genome Browser, can help to verify primer specificity against the reference genome or transcriptome.

Ø Primer modifications: Various modifications can be made to the primer sequences to improve their binding efficiencies, such as adding a GC clamp, a tail, or a tag. A GC clamp refers to adding a GC-rich sequence at the 3' end of the primer, which enhances the binding stability and specificity. A tail is a non-specific sequence added to the 5' or 3' end of the primer, which can aid in sequencing or multiplex PCR. A tag is a unique sequence added to the primer, which allows for the detection or purification of the amplified product.

In summary, the design of effective primers is crucial for the success of PCR, and several factors need to be considered and optimised, such as primer length, Tm, GC content, specificity, and modifications. Several online tools and software are available to facilitate primer design and optimization, such as OligoCalc, Primer3, IDT OligoAnalyzer, PrimerQuest, and others (Dieffenbach & Dveksler, 2003).

1.5.1.         Template quality and quantity

The quality and quantity of the DNA template used in a PCR reaction can significantly affect the outcome of the reaction. If the DNA template is not of good quality or is not present in sufficient quantity, the PCR reaction may fail, or the results obtained may be unreliable. Template quality can be affected by various factors, including DNA degradation, contamination, and modifications. Therefore, it is essential to ensure that the DNA template used for PCR is of good quality and is free from any contaminants that may interfere with the reaction. Several methods can be used to extract high-quality DNA from different sources, and the choice of method will depend on the type of sample being used (Murphy & Bustin, 2009). Template quantity is also critical in PCR, as too little DNA template can result in poor or no amplification, while too much DNA template can lead to nonspecific amplification and the production of unwanted products. The optimal amount of DNA template for PCR can vary depending on the type of sample and the PCR assay being used. Therefore, it is crucial to optimize the amount of DNA template used for PCR to achieve the best results.  Various methods can be used to measure the quantity and quality of DNA templates, including spectrophotometry, gel electrophoresis, and fluorescent dyes. Quantitative PCR (qPCR) is also a powerful tool that can be used to accurately quantify DNA template concentrations and assess the quality of the DNA.

In summary, the quality and quantity of the DNA template used in PCR reactions are essential factors that can significantly impact the success of the reaction. Therefore, it is crucial to ensure that high-quality DNA is used and that the appropriate amount of DNA template is optimized to achieve the best results (Demeke & Jenkins, 2010).

1.5.2.         MgCl2 concentration

Magnesium chloride (MgCl2) is a crucial component of PCR reactions, as it is required for the activity of the DNA polymerase enzyme. MgCl2 acts as a cofactor for the polymerase enzyme, helping to stabilise the enzyme-DNA complex and facilitating the correct alignment of the nucleotides during DNA synthesis. Therefore, the concentration of MgCl2 in the PCR reaction can significantly affect the efficiency and specificity of the reaction.  The optimal MgCl2 concentration for PCR can vary depending on several factors, including the type of DNA polymerase, used, the length and complexity of the template DNA, and the primer annealing temperature. Generally, the optimal MgCl2 concentration falls between 1.5 and 4 mM for most PCR reactions. However, it is essential to optimise the MgCl2 concentration for each specific PCR reaction to achieve the best results. Several strategies can be used to optimise the MgCl2 concentration in PCR reactions, including the use of gradient PCR, which involves testing a range of MgCl2 concentrations in the same reaction, and the use of MgCl2 titration, which involves testing a series of reactions with varying MgCl2 concentrations to determine the optimal concentration for the reaction.  It is also essential to note that high concentrations of MgCl2 can lead to nonspecific amplification, while low concentrations can result in poor amplification or no amplification at all. Therefore, it is crucial to carefully optimize the MgCl2 concentration for each PCR reaction to achieve the best results.

In summary, the MgCl2 concentration is a critical factor in PCR reactions, as it is required for the activity of the DNA polymerase enzyme. Therefore, it is essential to carefully optimise the MgCl2 concentration for each PCR reaction to achieve the best results (Bartlett & Stirling, 2003).

1.5.3.         Annealing temperature

The annealing temperature is a critical parameter in PCR, as it determines the specificity and efficiency of primer binding to the target DNA template during the annealing step of each cycle. The annealing temperature should be optimized for each primer set and DNA template to ensure successful amplification of the desired target sequence.  The optimal annealing temperature is influenced by several factors, including the length and composition of the primers, the GC content of the target DNA sequence, and the MgCl2 concentration in the reaction. Generally, the annealing temperature should be about 5°C below the melting temperature (Tm) of the primers, which is the temperature at which 50% of the primer molecules are annealed to the target DNA template. If the annealing temperature is too low, non-specific binding of primers to non-target regions of the template can occur, leading to the generation of unwanted PCR products or a decrease in amplification efficiency. On the other hand, if the annealing temperature is too high, the primers may not bind specifically to the target template, resulting in poor amplification efficiency (Rychlik et al., 1990).

1.5.4.         Cycling conditions

PCR cycling conditions refer to the temperature and time profile used during the three steps of the PCR cycle (denaturation, annealing, and extension) that are repeated multiple times to amplify the target DNA sequence. The cycling conditions must be Optimized to ensure efficient and specific amplification of the target DNA sequence while minimizing non-specific amplification and the formation of undesirable side products

The temperature and time for each step of the PCR cycle are determined by the melting temperature (Tm) of the primers and the optimal temperature for the activity of the DNA polymerase used. Typically, the cycling conditions involve an initial denaturation step at 95-98°C for 2-5 minutes, followed by 25-35 cycles of denaturation at 95-98°C for 10-30 seconds, annealing at 50-65°C for 10-30 seconds, and extension at 68-72°C for 30-90 seconds per kilobase of target DNA. A final extension step of 5-10 minutes at 68-72°C is usually performed to ensure the complete extension of all amplicons.  The annealing temperature is a critical factor in PCR optimization, as it determines the specificity and efficiency of primer annealing to the template DNA. The annealing temperature should be set above the Tm of the primers, but not too high to prevent non-specific annealing. A gradient PCR, where the annealing temperature is gradually increased across a range of values, can be used to determine the optimal annealing temperature for a specific primer pair. Other factors that can affect the cycling conditions in PCR include the GC content of the template DNA and the length and concentration of the primers. Optimization of these factors can also help to improve the efficiency and specificity of PCR amplification. Optimising the cycling conditions in PCR is a crucial step in achieving accurate and reliable amplification of the target DNA sequence. By carefully adjusting the temperature and time parameters, PCR can be used to amplify specific DNA sequences from a variety of sources and for various applications, including research and diagnostic purposes (Wu et al., 2011).

1.5.5.         Taq polymerase concentration

The concentration of Taq polymerase, an enzyme used in PCR, can have a significant impact on the efficiency and specificity of the reaction. Generally, the concentration of Taq polymerase used in PCR reactions is in the range of 0.025 to 0.05 units/μl. If the concentration of Taq polymerase is too low, the reaction may not amplify the desired product efficiently or at all. On the other hand, if the concentration is too high, it can result in the generation of non-specific products and the depletion of dNTPs, which are essential for the reaction.  Therefore, it is essential to Optimise the concentration of Taq polymerase for each PCR reaction to achieve optimal results. Several factors can affect the optimal concentration of Taq polymerase, including the length and complexity of the target DNA, the type of Taq polymerase used, and the other components of the reaction mixture. It is recommended to perform a gradient PCR to determine the optimal concentration of Taq polymerase for a specific PCR reaction. In gradient PCR, the concentration of Taq polymerase is varied in different reaction tubes, while keeping the other components of the reaction constant. The tubes are then run in the same thermal cycling program, and the optimal Taq polymerase concentration is determined by comparing the results obtained from each tube (Bustin et al., 2010).

1.5.6.         Buffer composition

PCR buffer composition is an important factor in Optimising the PCR reaction. PCR buffers contain a variety of components that help to maintain the pH, provide cofactors required for enzymatic activity, and facilitate the binding of primers to the template DNA. The major components of a typical PCR buffer include Tris-HCl, KCl, MgCl2, and sometimes betaine or DMSO. Tris-HCl serves as a buffer to maintain a constant pH during the reaction, while KCl helps to stabilize the DNA duplex by reducing electrostatic repulsion between negatively charged phosphate groups. MgCl2 is a cofactor for the Taq polymerase enzyme, which is necessary for enzymatic activity.   The concentration of each component in the buffer can be optimised to improve PCR performance. For example, the MgCl2 concentration can be titrated to achieve the optimal concentration for the specific Taq polymerase used in the reaction. Betaine or DMSO can be added to the buffer to improve the amplification of difficult templates or to reduce the formation of secondary structures in the template DNA. It is important to note that different PCR buffers may have different compositions, depending on the specific application or manufacturer. Therefore, it is recommended to follow the manufacturer's instructions when selecting and preparing a PCR buffer.

PCR optimization is a critical step to achieve accurate and reproducible results in PCR-based assays. By adjusting various parameters in the reaction, researchers can improve the sensitivity, specificity, and efficiency of PCR and obtain reliable data (Radstrom, et al., 2004).

1.6.      PCR Troubleshooting

PCR can be a powerful tool in molecular biology, but like any experimental technique, it can be prone to errors and issues (Huggett et al., 2015). Here are some common problems encountered in PCR and potential solutions:

1.6.1           Low yield or no amplification: When no amplification or low yield is observed in PCR, it could be due to several reasons. Here are some common troubleshooting steps:  

1.6.1.1      Template issues: Insufficient or poor-quality template DNA can result in no amplification or low yield. Try increasing the amount of template DNA or improving its quality through better extraction methods.

1.6.1.2      Primer issues: Poor primer design can lead to no amplification or low yield. Ensure that the primers are specific, have appropriate annealing temperatures, and are free from secondary structures. You can use software such as Primer3 or OligoCalc to design primers (Araujo & Franco 2019).

1.6.1.3      Annealing temperature: If the annealing temperature is too high, there might be insufficient annealing of primers resulting in no amplification or low yield. Lower the annealing temperature in increments of 1-2°C and Optimise the conditions to find the optimal temperature.

1.6.1.4      MgCl2 concentration: The MgCl2 concentration is critical for the activity of Taq polymerase. Insufficient or excessive concentrations of MgCl2 can result in no amplification or low yield. Optimise the MgCl2 concentration by trying different concentrations ranging from 1-4 mM.

1.6.1.5      Taq polymerase concentration: Insufficient Taq polymerase concentration can lead to no amplification or low yield. Optimize the Taq polymerase concentration by trying different concentrations ranging from 0.025-0.1 U/µl.

1.6.1.6      Cycling conditions: Cycling conditions such as denaturation and annealing times can affect the yield of PCR. Optimize these conditions to find the optimal times for each step.

1.6.1.7      Contamination: Contamination with PCR inhibitors such as salts, proteins, detergents, or PCR products can lead to no amplification or low yield. Ensure that your workspace is clean, and use new pipette tips, and aliquot reagents to prevent contamination.

1.6.1.8      Non-specific amplification: Non-specific amplification in PCR refers to the amplification of unintended DNA fragments that are similar in sequence to the target DNA. This can occur due to various reasons, such as the presence of non-specific binding sites, mispriming, or inadequate annealing conditions (Roux, 2009).

1.6.2           Strategies to troubleshoot and fix non-specific amplification in PCR:

1.6.2.1      Optimise annealing conditions: Non-specific amplification can occur if the annealing temperature is too low or too high, resulting in the binding of primers to non-specific sites. To Optimise annealing conditions, try a range of annealing temperatures in increments of 2-3°C to determine the optimal temperature for your primers and template (Schosk et al., 2003).

1.6.2.2      Optimise primer concentration: The concentration of primers can affect the specificity of amplification. Too low primer concentration can lead to non-specific amplification, while too high primer concentration can result in primer-dimer formation. Optimise primer concentration by testing a range of concentrations to determine the optimal concentration for your specific PCR reaction.

1.6.2.3      Use hot-start PCR: Hot-start PCR can prevent non-specific amplification by minimising primer-dimer formation and non-specific binding during the initial stages of the reaction. In hot-start PCR, Taq polymerase activity is blocked until the reaction reaches the annealing step, preventing the amplification of non-specific products.

1.6.2.4      Increase annealing time: Extending the annealing time can promote the binding of primers to specific sites on the template, reducing non-specific amplification. Try increasing the annealing time by 5-10 seconds to Optimize the annealing conditions.

1.6.2.5      Use a higher stringency buffer: Increasing the salt concentration in the PCR buffer can help to increase the stringency of the reaction, promoting specific primer-template binding and reducing non-specific amplification.

1.6.2.6      Design-specific primers: Non-specific amplification can be caused by primer sequences that have significant homology to non-target sequences. To prevent this, design primers with high specificity that only anneal to the target DNA sequence.

1.6.2.7      Reduce template DNA concentration: Non-specific amplification can occur due to high template DNA concentrations. Try reducing the template DNA concentration to Optimise the specificity of amplification.

1.6.2.8      Primer-dimer formation: PCR is a powerful molecular biology technique used to amplify a specific DNA sequence. However, one common problem encountered during PCR is the formation of primer dimers, which can interfere with the amplification of the desired DNA fragment. Primer-dimers are formed when two primers anneal to each other instead of binding to the target DNA sequence. This results in the formation of a non-specific product, which can reduce the yield of the desired product and complicate downstream analysis (Brownie et al., 1997).

Figure 1: PCR Components and Cycle

1.6.3           There are several steps that can be taken to troubleshoot primer-dimer formation in PCR:

1.6.3.1      Designing appropriate primers: Proper primer design is the key to minimizing primer-dimer formation. Primers should be designed to have melting temperatures (Tm) of around 55-65°C, with a maximum difference of 2°C between the two primers. The length of the primers should be between 18-22 nucleotides, with a GC content of 40-60% (Kalendar et al., 2009).

1.6.3.2      Adjusting annealing temperature: Adjusting the annealing temperature can help to minimise primer-dimer formation. Increasing the annealing temperature by 2-3°C above the Tm of the primers can reduce non-specific binding and primer-dimer formation.

1.6.3.3      Optimization of MgCl2 concentration: MgCl2 is an essential cofactor for PCR and plays a crucial role in primer annealing and polymerase activity. However, excessive or inadequate MgCl2 concentration can cause non-specific amplification, including primer-dimer formation. Optimising the MgCl2 concentration is important to minimise primer-dimer formation.

1.6.3.4      Using hot-start PCR: Hot-start PCR is a modification of the standard PCR protocol that can help to minimise primer-dimer formation. In hot-start PCR, the reaction is initially heated to a high temperature to activate the Taq polymerase while keeping the primers and other components inactive. This reduces the likelihood of primer-dimer formation.

1.6.3.5      Gel electrophoresis: Gel electrophoresis is a technique used to visualise PCR products and can be used to detect primer-dimer formation. A primer-dimer band will typically be smaller in size than the desired product and can be easily distinguished from the desired product on an agarose gel (Ririe et al., 1997)

In summary, minimising primer-dimer formation in PCR requires careful primer design, optimization of PCR conditions such as annealing temperature and MgCl2 concentration, use of hot-start PCR, and gel electrophoresis to detect primer-dimer formation. By carefully Optimising these parameters, primer-dimer formation can be minimised, and the yield and specificity of the desired PCR product can be improved.

1.6.4 High background: High background in PCR refers to the presence of non-specific bands or smears in the PCR product, which can interfere with the detection and analysis of the target DNA fragment. High background can be caused by a variety of factors, including contamination, incorrect primer design, and suboptimal PCR conditions. Below are some steps for troubleshooting high background in PCR:

1.6.4.1      Contamination: Contamination can occur at any stage of the PCR process and can result in high background. Possible sources of contamination include DNA, primers, Taq polymerase, buffers, and lab equipment. To troubleshoot contamination, it is recommended to:

●     Use fresh reagents and DNA samples

●     Use separate lab areas and pipettes for sample preparation, reaction setup, and post-PCR analysis

●     Use filter tips or change pipette tips between each step.

●     Use UV irradiation or other decontamination methods to clean work surfaces and equipment (Hoshino & Morimoto, 2008).

1.6.4.2      Primer design: Incorrect primer design can lead to non-specific amplification and high background. Primers should be designed to be specific to the target DNA sequence and avoid potential secondary structures, such as hairpins, self-dimers, and cross-dimers. It is recommended to:

●     Use primer design software to Optimise primer sequences

●     Check for potential primer-dimer formation or nonspecific binding using online tools, such as OligoCalc or Primer-BLAST

●     Check the specificity of the primers using BLAST or other sequence alignment tools (Brandes et al., 2007).

1.6.4.3      PCR conditions: Suboptimal PCR conditions can lead to a high background due to nonspecific amplification or primer-dimer formation. PCR conditions that can be Optimised include-

Annealing temperature: Adjust the annealing temperature to Optimise the specificity of the primers and minimise nonspecific binding.

Extension time: Shorten the extension time to minimise nonspecific amplification

MgCl2 concentration: Optimise the MgCl2 concentration to achieve optimal primer annealing and polymerase activity.

Template DNA concentration: Optimise the template DNA concentration to achieve optimal amplification without overloading the reaction.

Gel electrophoresis: High background can be visualised using gel electrophoresis, and it is recommended to troubleshoot high background using this technique. Possible solutions include:

●     Increase the annealing temperature or shorten the extension time to Optimise PCR conditions

●     Use a higher annealing temperature or lower primer concentration to reduce nonspecific binding

●     Use a nested PCR or touchdown PCR to increase the specificity of the amplification (Patton, 2000).

In conclusion, high background in PCR can be caused by contamination, incorrect primer design, and suboptimal PCR conditions. By troubleshooting these factors, it is possible to reduce the high background and improve the specificity and yield of the PCR product.

1.6.4.4 PCR inhibition: PCR inhibition refers to the presence of substances in the PCR reaction that interfere with the amplification of the target DNA fragment, resulting in a decreased yield or complete absence of PCR product. PCR inhibition can be caused by a variety of factors, including contaminants, inhibitors in the DNA sample, or the presence of inhibitors in the PCR reagents. Below are some steps for troubleshooting PCR inhibition:

Contamination: Contamination can occur at any stage of the PCR process and can result in PCR inhibition. Possible sources of contamination include DNA, primers, Taq polymerase, buffers, and lab equipment. To troubleshoot contamination, it is recommended to:

●     Use fresh reagents and DNA samples

●     Use separate lab areas and pipettes for sample preparation, reaction setup, and post-PCR analysis

●     Use filter tips or change pipette tips between each step

●     Use UV irradiation or other decontamination methods to clean work surfaces and equipment (Corless, 2010).

Inhibitors in the DNA sample: DNA samples can contain substances that inhibit PCR amplification, such as salts, organic compounds, and humic acids. To troubleshoot inhibitors in the DNA sample, it is recommended to:

●     Dilute the DNA sample to reduce the concentration of inhibitors

●     Use different DNA extraction methods, such as phenol-chloroform extraction or silica spin columns, to remove inhibitors

●     Add PCR enhancers, such as BSA or betaine, to reduce inhibition

Inhibitors in the PCR reagents: The PCR reagents themselves can also contain substances that inhibit PCR amplification, such as residual salts, EDTA, or detergents. To troubleshoot inhibitors in the PCR reagents, it is recommended to:

●     Use fresh PCR reagents, especially Taq polymerase, and buffer

●     Use water that is free of inhibitors, such as molecular biology-grade water or nuclease-free water

●     Adjust the pH of the reaction mixture to reduce inhibition

Template DNA concentration: The concentration of template DNA can also affect PCR amplification. Too much or too little template DNA can lead to PCR inhibition. To troubleshoot template DNA concentration, it is recommended to:

●     Optimise the template DNA concentration by testing different amounts of DNA

●     Dilute the DNA sample to reduce the concentration of inhibitors while maintaining a suitable amount of template DNA

PCR conditions: Suboptimal PCR conditions can also lead to PCR inhibition. PCR conditions that can be optimized include:

●     Annealing temperature: Adjust the annealing temperature to Optimise the specificity of the primers and minimise nonspecific binding

●     Extension time: Adjust the extension time to ensure that the DNA fragment is fully amplified without introducing PCR inhibitors

●     MgCl2 concentration: Optimise the MgCl2 concentration to achieve optimal primer annealing and polymerase activity (Malorny et al., 2004).

In conclusion, PCR inhibition can be caused by contamination, inhibitors in the DNA sample or PCR reagents, template DNA concentration, or suboptimal PCR conditions. By troubleshooting these factors, it is possible to reduce PCR inhibition and improve the yield and specificity of the PCR product (Demeke and Jenkins, 2010).

Conclusion

In conclusion, PCR has revolutionised the field of molecular biology since its development in the 1980s. From its humble beginnings as a technique to amplify DNA sequences, it has become a cornerstone of numerous research and clinical applications. The ability to amplify DNA from even minute samples has allowed for advances in fields such as diagnostics, genotyping, forensics, cancer research, and environmental monitoring, to name a few. Over the years, PCR has evolved to become faster, more sensitive, and more specific, with the development of new technologies such as qPCR and digital PCR. Despite the many advances in PCR technology, it is important to remember the fundamental principles of the technique and to optimise reactions carefully to achieve the best possible results. As PCR continues to be refined and expanded, it will undoubtedly remain a vital tool for researchers and clinicians alike.

Acknowledgement

The authors are also thankful to the Head, School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur for providing facilities and conducting the study. All the images were created by BioRender (https://www.biorender.com/).

Conflict of Interest

The authors had no conflict of interest.

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