Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a technique by which copies of a specific DNA strand are produced. These copies can subsequently, according to need, be used for both research and practical purposes. Polymerase chain reaction is a tool that is helping researchers and scientists across the globe in various scientific realms especially forensic science and medical diagnostics. This article discusses the key components of PCR including DNA templates, primers, dNTPs, types of PCR used in various industries, and also, its applications in forensic analysis, disease diagnosis, and pathogen detection.

What is Polymerase Chain Reaction?

Polymerase Chain Reaction (PCR) is a tool advancing our understanding across various scientific domains. Polymerase chain reaction is a technique in molecular biology that is used to make copies (amplify) of target DNA sequences quickly and with high accuracy. This technique was developed by an American biochemist, Kary Mullis, in 1983. PCR is a valuable tool and has applications in various fields, including medicine, biotechnology, forensic science, agriculture, and environmental science, among others.

Components of Polymerase Chain Reaction

Polymerase chain reaction uses some essential components that aid in the amplification of DNA sequences. These components include:

DNA Template

This is the DNA sample containing the specific sequence that is to be amplified. Without this target sequence, PCR cannot be performed.

Primer

These are short, single-stranded DNA sequences (usually ranging from 18 to 30 nucleotides in length) that are complementary to the DNA sequences. Primers bind to their complementary sequences on the DNA template and are then elongated along the target sequence by the DNA polymerase enzyme.

DNA Polymerase

This enzyme is responsible for the synthesis of a new DNA strand by adding complementary nucleotides to the target template during the extension phase of PCR. Taq polymerase is the most common type of DNA polymerase that is used in PCR, which is derived from a heat-stable bacterium Thermus aquaticus.

dNTPs (Deoxynucleotide Triphosphates)

These are the nucleotides required for synthesizing the new DNA strand. They include adenine, thymine, cytosine, and guanine.

Buffer Solution

PCR requires optimal pH and ionic conditions, for this a buffer solution is used. It maintains a stable environment for DNA polymerase enzyme to function.

Magnesium Ions (Mg²⁺)

Magnesium ions are essential cofactors that aid the DNA polymerase enzyme. They can be a part of the buffer solution or added separately to ensure efficient DNA synthesis.

Thermal Cycler

This laboratory instrument controls and automates the temperature cycles required for the denaturation, annealing, and extension phases of PCR.

PCR Plates

These are specialized plates that hold the PCR reaction mixture, including the DNA template, primers, DNA polymerase, dNTPs, buffer, and magnesium ions. They can withstand high-temperature changes during the PCR process.

Steps of Polymerase Chain Reaction

The steps involved in PCR include:

Step 1: Denaturation

In this step, the DNA sample with the target sequence is typically heated to temperatures between 94–98°C. This high temperature breaks the hydrogen bond between the double-stranded DNA, leading the DNA strands to separate or denature into two individual strands.

Step 2: Annealing

Following denaturation, the temperature is reduced to approximately 50–65°C. During this step, primers are introduced. These short DNA sequences attach to particular sections of the target DNA sequence, matching their sequence with the single-stranded target DNA template.

Step 3: Elongation

After primer annealing, the temperature is raised to around 72°C, allowing DNA polymerase to synthesize a new DNA strand by incorporating nucleotides that match the template strand. By extending from the primers, this enzyme builds a new complementary DNA strand, thus replicating the targeted DNA sequence.

By repeating these temperature cycles about 20-40 times, in the thermocycler, the amount of the target DNA doubles with each cycle, leading to an exponential increase in the number of copies. As a result, even a short target DNA sequence can be amplified to millions or billions of copies within a short time.

Types of Polymerase Chain Reaction

There are various types of PCR used in molecular biology, these include:

Real-time PCR

Also referred to as quantitative PCR (qPCR), real-time PCR allows for the simultaneous amplification and measurement of DNA in real-time. This is accomplished using fluorescent dyes or probes that produce fluorescence during the DNA amplification process.

Nested PCR

Nested PCR is much more sensitive and specific than traditional PCR as it uses dual primer sets. These dual primer sets are used for two successive PCR reactions. The first set of primers known as external primers are used in the first PCR that amplifies a larger initial DNA fragment. Then, a second PCR known as nested PCR uses a set of internal primers that are designed to anneal within the region amplified by the first PCR.

Multiplex PCR

This type of PCR allows for multiple target DNA sequences to be simultaneously amplified by a single PCR reaction. The specificity and efficiency of multiplex PCR rely on the precise design of primer sets to ensure specific amplification of each target sequence.

Hot Start PCR

This technique uses modified DNA polymerases that remain inactive at lower temperatures, preventing nonspecific amplification and enhancing the specificity and efficiency of PCR reactions.

Applications of Polymerase Chain Reaction

Polymerase chain reaction (PCR) has a wide range of applications including:

Forensic Analysis

In forensic science, PCR is mostly used for DNA profiling. For analysis and identification, even a tiny amount of DNA from a crime scene can prove useful. The DNA can be amplified using PCR to provide enough material for investigation.

Genetic Testing and Diagnosis

PCR is a great tool for the identification of genetic diseases. It amplifies the specific DNA sequence associated with the particular genetic disorder thus aiding in early diagnosis and treatment planning.

Infectious Disease Detection

During COVID-19, PCR served as a crucial technique that was used to detect the presence of pathogens like viruses and bacteria in clinical samples. It can also be used for diagnosing other diseases including HIV, and tuberculosis, amongst others.

Paternity and Relationship Testing

To determine paternity and other familial relationships with precision, PCR-based DNA tests are used. It compares specific genetic markers between individuals to determine the relationship.

Food Safety

For, food safety and quality control, PCR is used to efficiently detect pathogens or genetically modified organisms (GMOs) in food products.

Conclusion – Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a transformative technique that is driving advancements across various scientific disciplines. PCR’s exponential amplification capability has revolutionized research methodologies, and applications ranging from forensic analysis to agriculture. As we deal with the challenges of today’s science and healthcare, PCR’s lasting importance shows how essential it is for enhancing diagnostics, and advancements that benefit humanity.

FAQs on Polymerase Chain Reaction (PCR)

What is the Polymerase Chain Reaction Process?

Polymerase Chain Reaction is a technique used to amplify and make copies of a target DNA sequence. This technique involves three main steps- denaturation, annealing, and elongation.

What are the 4 Steps of PCR?

The four steps of PCR include- collection, preparation, amplification, and post-PCR clean-up.

What is the Principle of PCR?

PCR is based on the following principle- the ability to amplify or make copies of target segments of DNA, exponentially and accurately.

What are the Four Important Applications of PCR?

The four important applications of PCR include genetic engineering, diagnostics, forensic analysis, and DNA isolation.

What PCR is used for?

PCR is used for amplifying target DNA sequences, and has applications in gene cloning, disease diagnosis, forensic DNA analysis, and other scientific fields.

What is the Main Purpose of PCR?

The highly sensitive PCR method enables the quick amplification of a particular DNA segment.

What is Role of Primers in PCR?

By hybridizing with the sample DNA, two primers in the PCR process identify the region that will be amplified.