INTRODUCTION:

Polymerase Chain Reaction PCR is a widely used molecular biology technique that amplifies DNA sequences in vitro, making millions or billions of copies from just a few starting copies.

PCR involves a series of temperature changes to initiate DNA denaturation, primer annealing, and DNA synthesis using a thermostable DNA polymerase. This technique has revolutionized genetic research, clinical diagnostics, forensic science, and many other fields by enabling rapid and highly sensitive detection and quantification of specific DNA sequences. PCR is a critical tool for various applications, including gene expression analysis, genetic testing, infectious disease diagnosis, and DNA fingerprinting.


Table of Content:

History of PCR:

PCR, short for Polymerase Chain Reaction, was first developed in the early 1980s by Kary Mullis, an American biochemist. Mullis' invention of PCR was a breakthrough in molecular biology, revolutionizing the way scientists study and manipulate DNA.

Mullis initially conceived of the idea of PCR while driving down a California highway in 1983, realizing that he could use a pair of primers to amplify specific DNA sequences. He quickly developed the method and published his findings in 1985. PCR's potential quickly became apparent, and the technique quickly gained popularity in the scientific community. In 1993, Mullis and his colleagues were awarded the Nobel Prize in Chemistry for their invention of PCR, recognizing the enormous impact that the technique has had on biomedical research and diagnostics.

Since its inception, PCR has undergone many advancements and modifications, including the development of quantitative PCR (qPCR), digital PCR (dPCR), and reverse transcription PCR (RT-PCR). These variations have expanded PCR's applications, making it a versatile and powerful tool in fields such as medicine, genetics, forensics, and agriculture.


How does PCR work?

PCR is based on a simple concept making multiple copies of a specific DNA segment. This is achieved by using a DNA polymerase enzyme that can add nucleotides to a single-stranded DNA template. The polymerase is activated by heating the sample to around 95°C, which separates the two strands of DNA. Once the strands are separated, the temperature is lowered to around 50-60°C, which allows short DNA primers to bind to the template.

These primers are designed to be complementary to the ends of the target DNA sequence. Once the primers are in place, the temperature is raised to around 72°C, which is the optimal temperature for the polymerase to start adding nucleotides. The polymerase adds nucleotides to the DNA template, extending the length of the strand.

This process continues until the polymerase reaches the end of the template or another barrier, such as a primer or enzyme blocker. This creates two copies of the target DNA sequence, which can then be used as templates for further amplification.

PCR can be used for a variety of applications, including genetic testing, gene expression analysis, and DNA sequencing. It has revolutionized the field of molecular biology by allowing researchers to quickly and easily amplify specific DNA sequences, even from very small samples.



Steps of PCR

There are three main stages in a PCR

 (Polymerase Chain Reaction) cycle:

  • Denaturation
  • Annealing
  • Extension

What happened at Denature stage?


Denaturation is the first stage of PCR, which involves the separation of the two complementary strands of the DNA double helix. During this process, the double-stranded DNA is exposed to high temperatures, typically between 94-98°C, for a short period of time, usually around 30 seconds to 1 minute.

This temperature causes the hydrogen bonds between the base pairs to break, resulting in the separation of the two DNA strands. The denaturation stage is a crucial step in PCR because it allows the primers to bind to the single-stranded DNA template in the next stage, Annealing. Without proper denaturation, the primers cannot bind to the template DNA, and the PCR reaction will not work efficiently.

In summary, the denaturation stage of PCR involves exposing the DNA to high temperatures, typically between 94-98°C, for a short period of time, usually around 30 seconds to 1 minute. This process separates the two complementary strands of the DNA double helix, allowing the primers to bind to the single-stranded DNA template in the next stage, Annealing. Proper denaturation is essential for the success of the PCR reaction.


What happened at Annealing stage?


During the annealing stage in PCR (polymerase chain reaction), the temperature of the reaction mixture is lowered to allow the primers to anneal, or bind, to the single-stranded DNA template. This is a critical step in the PCR process because it determines where the DNA polymerase will begin synthesizing new strands of DNA.

The annealing temperature used in PCR is typically a few degrees below the melting temperature (Tm) of the primers, which is the temperature at which half of the primers are annealed to the template DNA and half are not. By using a lower temperature, the primers can bind specifically and efficiently to the complementary sequences on the template DNA.

The annealing stage typically lasts for 20-30 seconds, depending on the specific primers and template being used. After annealing, the temperature is raised again to the extension temperature, which allows the DNA polymerase to begin synthesizing new strands of DNA.

What happened at extension stage?


During the extension stage of PCR (Polymerase Chain Reaction), the temperature is typically set between 68-72°C, and it is the second of the three steps in each PCR cycle. During this stage, the DNA polymerase enzyme adds nucleotides to the 3' end of the primers, extending the new DNA strand in the 5' to 3' direction. The extension time depends on the length of the amplicon (the DNA sequence being amplified), and the temperature and fidelity of the polymerase used.

Typically, the extension time is approximately 30 seconds per kilo base of DNA being amplified. For example, if the target sequence is 1 kilo base in length, the extension time would be around 30 seconds. During the extension stage, the primers annealed to the DNA template serve as the starting point for the synthesis of new strands of DNA. The DNA polymerase enzyme adds complementary nucleotides to the 3' end of the primers. The nucleotides are added one at a time, and the polymerase enzyme moves along the DNA template strand in a 5' to 3' direction, continuously adding new nucleotides.

The extension stage is critical for the success of PCR because it ensures that the newly synthesized strands are completely copied from the original DNA template. This stage is also responsible for amplifying the DNA target sequence, increasing the amount of the target sequence by two-fold with each PCR cycle. Once the extension stage is complete, the cycle is repeated from the denaturation step, starting the next cycle of PCR amplification. The extension stage is a crucial step in the PCR process that allows for the rapid and efficient amplification of specific DNA sequences.


Polymerase Chain Reaction PCR is a widely used molecular biology technique that amplifies DNA sequences in vitro, making millions or billions of copies from just a few starting copies.

3 Stages of PCR



DNA Template:

The DNA template is the starting material for PCR. It contains the sequence of DNA that will be amplified.

DNTPs:

DNTPs (Deoxynucleoside triphosphates) are the building blocks of DNA. They are the individual units that are combined to form the DNA strands during PCR.

Taq Polymerase:

Taq polymerase is a heat-stable enzyme that is used to amplify the DNA template in PCR. It is derived from the bacterium Thermus aquaticus, which is found in hot springs. Taq polymerase is able to withstand the high temperatures that are used in the denaturation step of PCR, which makes it ideal for this application.

Primers:

Primers are short, synthetic pieces of DNA that are designed to anneal (bind) to specific sequences on the DNA template. They are the starting points for DNA synthesis during PCR, and they define the region of DNA that will be amplified. Primers are usually around 20-30 nucleotides in length and are designed to be complementary to the ends of the target sequence.


Applications of PCR:

The applications of PCR are vast, and this technique has revolutionized the way we study DNA. Here, we will explore some of the most common applications of PCR and how they are used in research.

Disease Diagnosis

PCR is widely used in disease diagnosis, particularly for infectious diseases. This technique can detect the presence of viral or bacterial DNA in patient samples, such as blood or saliva. PCR is also used to diagnose genetic disorders, such as cystic fibrosis or sickle cell anemia. This application of PCR has transformed the field of medicine, allowing doctors to diagnose diseases more accurately and quickly.

Forensic Science

PCR is an essential tool in forensic science, as it can be used to analyze DNA evidence left at crime scenes. This technique can amplify even the tiniest amounts of DNA, making it possible to identify suspects or victims. PCR can also be used to compare DNA samples from different sources, such as blood samples from a crime scene and a suspect.

Gene Expression Analysis

PCR is used to study gene expression, which is the process by which genes are used to make proteins. Scientists use PCR to measure the amount of RNA (a molecule that carries genetic information) produced by a particular gene. This information can help researchers understand how genes are regulated and how they contribute to specific diseases.

Environmental Monitoring

PCR is used to monitor environmental samples, such as soil or water, for the presence of microorganisms. This technique can detect bacteria, viruses, and fungi that may be harmful to human health or the environment. PCR is also used to study microbial communities and how they interact with their environment.

Food Safety

PCR is used in food safety to detect the presence of pathogens, such as Salmonella or E. coli, in food products. This technique can identify the source of contamination and prevent outbreaks of foodborne illness. PCR is also used to authenticate food products, such as identifying the species of fish in a fish fillet.

Drug Development

PCR is used in drug development to identify potential drug targets, screen drug candidates, and evaluate the efficacy of drugs. For example, PCR can be used to measure changes in gene expression in response to a particular drug, which can help identify the most effective treatments for various diseases.

Cancer Diagnosis & Treatment

PCR is used in cancer diagnosis and treatment to detect genetic mutations that are associated with cancer. By analyzing DNA samples from cancer patients, PCR can help doctors determine the best course of treatment for individual patients, such as selecting targeted therapies that are tailored to the patient's specific genetic profile. Infectious

Disease Diagnosis

PCR is used to diagnose infectious diseases by detecting the DNA of pathogens in patient samples, such as blood or urine. This technique can identify the specific pathogen causing the infection and can help doctors choose the most effective treatment for the patient.

Genetic Testing

PCR is used in genetic testing to detect mutations in genes that are associated with inherited diseases, such as cystic fibrosis or Huntington's disease. PCR can also be used to screen for genetic diseases in newborns, which can help doctors identify and treat these conditions early in life.

Quality Control

PCR is used in pharmaceutical manufacturing to ensure the quality of drug products. For example, PCR can be used to detect the presence of contaminants in raw materials or finished products, such as bacteria or viruses. This technique can also be used to monitor the purity and potency of drugs throughout the manufacturing process.

In summary, PCR is an essential tool in the pharmaceutical and healthcare industries, with applications ranging from drug development and cancer treatment to infectious disease diagnosis and genetic testing. Its ability to detect specific DNA sequences has revolutionized the way we diagnose and treat diseases, and has contributed significantly to advances in medical research and technology.

Limitations Of PCR:

False Positives and Negatives

One of the significant limitations of PCR is the potential for false positives and negatives. This can occur due to various factors, such as contamination, primer mismatches, or inadequate sample quality. False positives can lead to incorrect diagnoses, while false negatives can result in missed diagnoses.

Inhibitors

PCR can be inhibited by substances present in the sample, such as blood, feces, or soil. These inhibitors can interfere with the amplification process, resulting in poor quality or failed PCR products. Inhibitors can be removed by various methods, such as DNA purification or dilution, but these steps can increase the risk of sample loss or contamination.

Limited DNA Sequence Amplification

PCR can only amplify DNA sequences that are flanked by primers. If the target DNA sequence is too long or complex, the primers may not be able to bind effectively, resulting in poor amplification or no amplification. In addition, PCR products may have limited sequence diversity, which can affect downstream applications, such as sequencing or cloning.

Specialized Equipment & Expertise

PCR requires specialized equipment, such as thermal cyclers and gel electrophoresis systems, and expertise in molecular biology techniques. The cost of equipment and reagents can be significant, particularly for high-throughput applications. In addition, PCR requires careful optimization and validation, which can be time-consuming and require significant expertise.

Limited Quantitative Information

PCR is a qualitative technique, which means that it can only detect the presence or absence of a specific DNA sequence. While PCR can provide semi-quantitative information, such as relative gene expression levels, it cannot provide absolute quantification without additional methods, such as digital PCR or qPCR.