UNIVERSITY OF LAHORE SARGODHA CAMPUS
(Department of zoology)
Assignment # 03
Polymerase Chain Reaction(PCR)
Course : Bioinformatics
Submitted To:
Prof. Muhammad Usman
Submitted By: Ali Kaishan
Reg #: 70163029
Semester: 7th
Submission Date: 09-12-2024
Polymerase Chain Reaction:
Introduction: Polymerase Chain Reaction (PCR) is a widely used
technique in molecular biology that allows for the amplification of
specific DNA sequences. This technique was developed by Kary Mullis
in 1983 and has since become a fundamental tool in genetic research,
medical diagnostics, forensic science, and biotechnology.
Principle of PCR: PCR is based on the enzymatic replication of DNA
in vitro. It utilizes DNA polymerase to amplify a targeted DNA segment
through a series of temperature-regulated steps. The technique relies on
the ability of complementary DNA strands to hybridize and the
enzyme’s capacity to extend the DNA sequence.
Components of PCR:
1. Template DNA: The DNA sample containing the target sequence
to be amplified.
2. Primers: Short single-stranded DNA sequences that flank the
target DNA region.
3. DNA Polymerase: A heat-stable enzyme, such as Taq polymerase,
that synthesizes new DNA strands.
4. Nucleotides (dNTPs): Building blocks for DNA synthesis.
5. Buffer Solution: Maintains optimal conditions for the reaction,
including pH and ion concentration.
Steps of PCR: PCR consists of three main steps, which are repeated in
cycles to exponentially amplify the target DNA sequence:
1. Denaturation (94-98°C): The double-stranded DNA is heated to
separate it into two single strands.
2. Annealing (50-65°C): Primers bind to their complementary
sequences on the single-stranded DNA.
3. Extension (72°C): DNA polymerase synthesizes new DNA
strands by adding dNTPs in the 5' to 3' direction.
These steps are repeated for 20-40 cycles, leading to the exponential
amplification of the target DNA sequence.
Types of PCR:
1. Conventional PCR: Standard PCR method used for DNA
amplification.
2. Real-Time PCR (qPCR): Monitors DNA amplification in real-
time using fluorescent dyes.
3. Reverse Transcription PCR (RT-PCR): Converts RNA into
DNA before amplification, commonly used for studying gene
expression.
4. Multiplex PCR: Uses multiple primer sets to amplify different
DNA regions simultaneously.
5. Nested PCR: Enhances specificity by using two sets of primers in
successive reactions.
6. Touchdown PCR: Reduces non-specific binding by gradually
lowering the annealing temperature.
Applications of PCR: PCR is widely used in various fields, including:
Medical Diagnostics: Detecting infectious diseases (e.g., COVID-
19, HIV), genetic disorders, and cancer mutations.
Forensic Science: DNA fingerprinting for crime scene
investigations.
Genetic Research: Studying gene expression, sequencing, and
genetic variation.
Biotechnology: Producing recombinant DNA and genetically
modified organisms (GMOs).
Agriculture: Identifying plant pathogens and improving crop
breeding techniques.
Advantages of PCR:
High sensitivity and specificity.
Rapid and efficient DNA amplification.
Requires minimal DNA sample.
Adaptable to various modifications for different applications.
Limitations of PCR:
Prone to contamination, leading to false-positive results.
Requires precise temperature control and expensive equipment.
Enzyme inhibitors can affect reaction efficiency.
Limited to relatively short DNA fragments.
Conclusion: PCR is an indispensable tool in molecular biology,
revolutionizing genetics, diagnostics, and forensic science. Despite its
limitations, advancements such as digital PCR and high-fidelity
polymerases continue to improve its accuracy and applicability. The
development of PCR-based techniques has paved the way for numerous
scientific and medical breakthroughs.
