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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

  1. Melting: Heating to 95°C breaks hydrogen bonds between DNA strands.

  2. Annealing: Cooling to 54°C allows primers to bind to specific DNA sequences.

  3. 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.