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DNA Replication Overview
Definition: DNA replication is the production of exact copies of DNA with identical base sequences.
Importance: Essential for reproduction, growth, and tissue replacement in multicellular organisms.
Creating Replica
Process: DNA replication creates new DNA strands that are identical in base sequences to existing strands.
Unlimited Replication: The structure of DNA allows for continuous replication over billions of years since the origin of life.
Biological Processes Involved in DNA Replication
Reproduction
Offspring require copies of parental base sequences; thus, DNA replication is vital for reproduction.
Growth and Tissue Replacement
Multicellular organisms need complete base sequences in each cell before cell division.
Necessary for growth and replacing worn-out cells, e.g., skin cells.
Semi-Conservative Nature of DNA Replication
Mechanism: During replication, the double helix separates, using each original strand as a template for new strand synthesis.
Replication Fork: The site of active DNA copying where new strands are formed progressively.
Complementary Base Pairing
Template Dependency: The base sequence on the template strand determines the base sequence of the new strand. Only complementary bases can form hydrogen bonds and attach.
Base Pairing Rules:
Adenine (A) pairs with Thymine (T).
Cytosine (C) pairs with Guanine (G).
Accuracy in DNA Replication
The rule of complementary base pairing is crucial for ensuring identical DNA molecules are produced.
High accuracy with only about 1 error per 10 billion bases, maintaining genetic integrity.
In humans, a diploid cell has approximately 6 billion base pairs, resulting in about 0.6 errors during replication.
Roles of Helicase and DNA Polymerase in DNA Replication
Replisome
Definition: A complex of proteins (replisome) including helicase and DNA polymerase essential for DNA replication.
Helicase
Function: Unwinds and separates the DNA strands by breaking hydrogen bonds between bases.
Analogy: Functions like a zip fastener, often referred to as unwinding and unzipping DNA.
Managing Tension: Prevents supercoiling by relieving tension during DNA unwinding.
DNA Polymerase
Function: Assembles new DNA strands using original strands as templates.
Process: Adds nucleotides one by one; nucleotides must pair correctly with the template strand.
Bond Formation: Links nucleotides by forming covalent bonds between phosphate and sugar of successive nucleotides.
Polymerase Chain Reaction (PCR)
Overview
Definition: PCR is an automated method for DNA replication, allowing amplification of specific DNA sequences.
Temperature Cycling: Consists of melting, annealing, and elongation phases facilitated by thermal cycling.
Steps in PCR Cycle
Melting: Heating to 95°C breaks hydrogen bonds between DNA strands.
Annealing: Cooling to 54°C allows primers to bind to specific DNA sequences.
Elongation: Heating to 72°C creates optimal conditions for Taq DNA polymerase to synthesize new strands.
Taq DNA Polymerase
Source: Derived from Thermus aquaticus, which thrives in high-temperature environments.
Efficiency: Capable of rapidly assembling DNA, essential for high-throughput PCR.
Gel Electrophoresis
Overview
Purpose: Used for separating DNA molecules by length following PCR amplification.
Process: DNA samples are loaded into wells in a gel and subjected to an electric field, causing smaller fragments to migrate further.
Visualization
Resulting Bands: DNA molecules form visible bands indicating their lengths. A DNA ladder is used for size estimation.
Applications of PCR and Gel Electrophoresis
Testing for Coronaviruses
Method: Involves swabbing for viral RNA, converting it to DNA, and using PCR for amplification and detection.
Monitoring: Fluorescent markers indicate positive results based on amplified DNA levels.
Advantages and Disadvantages
Advantages: Extremely sensitive and specific, enabling detection of low viral loads.
Disadvantages: Requires costly equipment and does not provide immediate results.
DNA Profiling for Paternity Testing
Technique: Uses short tandem repeats (STRs) to distinguish individuals based on pattern variations in repeats.
Procedure: Involves PCR amplification and gel electrophoresis to generate unique DNA profiles.