“Polymeric Chain Reaction (PCR): A Revolutionary Molecular Tool”

Polymerase Chain Reaction (PCR) is a widely used molecular biology technique that amplifies DNA (deoxyribonucleic acid) segments. It involves a repetitive cycle of three main steps: denaturation, where the DNA strands are separated by heating; annealing, where short DNA primers bind to the target sequences; and extension, where DNA polymerase synthesizes new strands. This process results in the exponential amplification of the target DNA, enabling its detection and analysis. PCR has numerous applications in genetics, forensics, diagnostics, and research.

Kary Mullis

Polymerase Chain Reaction (PCR) was developed by Kary B. Mullis in 1983, earning him the Nobel Prize in Chemistry in 1993. Mullis conceived the idea while driving and realized the potential of a process to exponentially amplify DNA. PCR revolutionized molecular biology by enabling the rapid and precise replication of DNA in vitro. Its impact has been profound, facilitating advancements in genetics, diagnostics, forensics, and various scientific fields. PCR has become a fundamental tool, empowering researchers and clinicians to study and manipulate DNA with unparalleled efficiency and accuracy.

PCR or Polymerase Chain Reaction is a technique used in molecular biology to create several copies of a certain DNA segment.The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesis new strands of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre-existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase.

There are three major steps :

The following are the components of PCR:

  • DNA Template – The DNA of interest from the sample.
  • DNA Polymerase – Taq Polymerase is used. It is thermostable and does not denature at very high temperatures.
  • Oligonucleotide Primers – These are the short stretches of single-stranded DNA complementary to the 3’ ends of sense and anti-sense strands.
  • Deoxyribonucleotide triphosphate – These provide energy for polymerization and are the building blocks for the synthesis of DNA. These are single units of bases.
  • Buffer System – Magnesium and Potassium provide optimum conditions for DNA denaturation and renaturation.

There are several types of Polymerase Chain Reaction (PCR), each designed for specific applications. Here are some common types along with their principles:

1. Conventional PCR:

  • Principle: Involves three main steps – denaturation, annealing, and extension – to amplify a specific DNA sequence.
  • Applications: Basic DNA amplification, cloning, and gene expression analysis.

2. Reverse Transcription PCR (RT-PCR):

  • Principle: Converts RNA into complementary DNA (cDNA) using reverse transcriptase before proceeding with the conventional PCR amplification.
  • Applications: Gene expression analysis, studying RNA viruses. 

3. Quantitative PCR (qPCR):

  • Principle: Measures the amount of PCR product in real-time as amplification occurs, allowing for quantification of the initial DNA template.
  • Applications: Gene expression quantification, viral load determination, and other quantitative analyses.

4. Nested PCR:

  • Principle: Involves two sets of primers – an outer and an inner pair. The first PCR amplifies a larger fragment, and a second PCR with the inner primers amplifies a smaller, nested fragment within the first product.
  • Applications: Increased specificity, reducing non-specific amplification. 

5. Multiplex PCR:

  • Principle: Amplifies multiple target sequences in a single reaction using multiple primer pairs.
  • Applications: Simultaneous detection of multiple genes or pathogens.

6. Digital PCR (dPCR):

  • Principle: Divides the PCR reaction into thousands of tiny partitions, allowing for absolute quantification of DNA by counting positive and negative partitions.
  • Applications: Extremely precise quantification, rare mutation detection.

7. In situ PCR:

  • Principle: Amplifies DNA directly within fixed cells or tissue sections.
  • Applications: Localization of specific DNA sequences within cells or tissues.

These variations in PCR techniques cater to diverse research and diagnostic needs, offering flexibility and specificity in studying and manipulating genetic material.

Polymerase Chain Reaction (PCR) has a wide range of applications across various scientific disciplines. Here are some key applications of PCR:

  • PCR is primarily used to amplify specific DNA sequences, making it a fundamental tool for cloning and further molecular biology studies.
  • PCR is extensively employed for the detection of pathogens, including bacteria, viruses, and fungi, aiding in the diagnosis of infectious diseases.
  • PCR is crucial for genetic testing, allowing the identification of specific genes associated with genetic disorders or predispositions.
  • PCR is used in forensic science to amplify DNA from crime scene samples, facilitating DNA profiling and identification of individuals.
  • PCR is often integrated into DNA sequencing workflows to amplify specific regions of interest before sequencing.
  • Reverse Transcription PCR (RT-PCR) is employed to study gene expression by amplifying complementary DNA (cDNA) from RNA samples.
  • PCR is utilized to identify specific DNA mutations, aiding in the diagnosis of genetic disorders or monitoring genetic changes in cancer.
  • PCR is crucial in studying the genetics of infectious agents, tracking outbreaks, and understanding the epidemiology of diseases.
  • PCR is applied in environmental science for the detection and monitoring of microbial populations in soil, water, and air.
  • PCR is used to detect foodborne pathogens, ensuring the safety of food products by identifying contamination.
  • PCR is involved in pharmacogenetic studies, helping to understand how genetic variations influence an individual’s response to drugs.
  • PCR is employed in the amplification of ancient DNA, allowing researchers to study genetic material from archaeological or paleontological samples.
  • PCR-based methods are used for non-invasive prenatal screening to detect genetic abnormalities in the fetus.
  • PCR is crucial in monitoring the viral load in individuals infected with viruses such as HIV or hepatitis, aiding in disease management.

2 thoughts on ““Polymeric Chain Reaction (PCR): A Revolutionary Molecular Tool””

Leave a Comment