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DNA Notes - Key Concepts (Exam Prep)

DNA as Inheritance Evidence

  • Evidence timeline for DNA as the genetic material
    • 1868: Friedrich Miescher isolates DNA (called "nuclein"); contains phosphorus but not sulfur. First isolation of DNA.
    • Early 1900s: Dyes stain DNA; more DNA → more dye. Flow cytometry shows cells with different DNA content; some cells have about twice as much DNA as others. Note: Proteins are also part of chromosomes.
  • Quick takeaway: These findings laid groundwork that DNA carries hereditary information, not proteins alone.

DNA Structure – Foundations Before 1950

  • DNA is a long polymer made of nucleotides; monomers are A, G, C, T.
  • Purines: A and G; Pyrimidines: C and T.
  • 3D structure and base-pairing rules were not yet known.

Chargaff's Rule and Base Pairing

  • Chargaff (1950) rules:
    • ext{%A} = ext{%T}
    • ext{%C} = ext{%G}
    • ext{%A} + ext{%G}
      eq ext{%C} + ext{%T}
  • Implication: Ratios of bases are specific, enabling the later discovery of base-pairing.

Base Pairing and Hydrogen Bonds

  • Purine always pairs with pyrimidine:
    • A ext{ pairs with } T ext{ (or U in RNA)} with 2 hydrogen bonds.
    • C ext{ pairs with } G with 3 hydrogen bonds.
  • Quick exercise: If guanine is 20 ext{%}, cytosine is 20 ext{%}; adenine and thymine are each 30 ext{%} (because A=T and C=G).

DNA Structure – Franklin & Crick

  • Rosalind Franklin (1952): X-ray crystallography showed DNA is long, helical, uniform in thickness; bases interior; backbone on the outside.
  • Watson & Crick (1953): Used Franklin’s data and Chargaff’s rules to build the DNA double-helix model; proposed base-pairing and antiparallel strands; published the structure.
  • Franklin died young; Watson-Crick model explained how DNA could function as genetic material.

DNA Structure – Function and Orientation

  • After identifying DNA as genetic material, two key questions:
    • How is DNA replicated between cell divisions?
    • How does DNA direct synthesis of specific proteins? (addressed in later chapters)
  • Key ideas: double-helix structure enables templated replication and information flow.

DNA Replication – Models and Evidence

  • Replication models proposed from the idea that each strand could be a template:
    • Semiconservative
    • Conservative
    • Dispersive
  • Meselson & Stahl (1958): used heavy isotopes to test models; results supported the semiconservative model.

DNA Replication – Semiconservative Evidence

  • If replication were conservative, or dispersive, testable patterns would emerge across generations; results matched semiconservative replication (one old strand + one new strand per daughter DNA).

DNA Replication – Process Overview

  • Overall steps: Initiation → Elongation → Termination.
  • Prokaryotes replicate much faster (≈ 1100 rac{bp}{s}; genome in ~40 ext{ minutes} for E. coli).
  • Eukaryotes replicate much slower (≈ 10 rac{bp}{s}).
  • Initiation involves origin recognition and assembly of replication machinery; replication forks form and proceed.

DNA Replication – Key Enzymes & Components

  • Unwinding and stabilization: DNA Helicase, Single-Stranded Binding Proteins (SSBs), Topoisomerase, Primase (RNA primer).
  • Elongation enzymes: DNA Polymerase III (main replicative polymerase in prokaryotes).
  • Processing & joining: DNA Polymerase I, DNA Ligase.
  • Primer: RNA primer provides starting point for DNA synthesis.

DNA Replication – Antiparallel Synthesis and Strand Fate

  • DNA is antiparallel; synthesis occurs in the 5′ → 3′ direction.
  • Leading strand: synthesized continuously toward the replication fork.
  • Lagging strand: synthesized discontinuously as Okazaki fragments away from the fork.

DNA Replication – More on the Enzymes

  • DNA Polymerase III extends new DNA strand.
  • DNA Polymerase I replaces RNA primers with DNA.
  • DNA Ligase seals nicks between Okazaki fragments.

Practice Concepts – Leading vs Lagging

  • Which strand is the leading strand? Typically the top (continuous) strand in the common schematic; the bottom strand is lagging (Okazaki fragments).

End Replication Problem & Telomeres

  • End replication problem: chromosomes shorten with each cell division.
  • Telomeres: repetitive end sequences that protect genes during replication.

DNA Mutations – Causes

  • Mutations arise from:
    • Replication error
    • Chemical changes
    • Chemical/environmental damage
    • Cell division errors

Mutation Types

  • Point mutation: insertion, deletion, or substitution of a single base pair.
  • Additional notes: Replication errors + spontaneous chemical changes account for a substantial portion of cancers (roughly rac{2}{3} of cases).

Mutagens & Repair

  • Mutagens: substances that chemically alter DNA and induce mutations.
  • Excision repair: removes and replaces damaged nucleotides; most DNA damage is repaired.

Chromosomal Mutations

  • Chromosomal mutations affect larger regions and multiple genes, not just a single base.

Mutation and Phenotype

  • DNA regions: Genetic, Regulatory, and Junk regions ( Satellites and Introns ).
  • If a mutation falls in junk regions, it can be silent; if it does not change an amino acid, it can be silent as well.

PCR – Polymerase Chain Reaction

  • Purpose: amplify millions of copies of a DNA fragment for analysis/manipulation.
  • Core cycle (repeated 35–40 times):
    • Melt: 95^{\circ}C for 30 ext{ s}
    • Anneal: 55^{\ }C for 30 ext{ s}
    • Extend: 72^{\circ}C for 1 ext{ min}
  • Components:
    • DNA template
    • A thermostable DNA polymerase (e.g., Taq)
    • Two short primers
    • Four dNTPs: dATP, dCTP, dGTP, dTTP
    • Salts and buffer

PCR – What Initiates Copying?

  • Why strands separate in the cell and what polymerase needs to start copying:
    • Strands separate at the replication fork; primers are required to start synthesis.
    • Primer orientation: the new strand is built in the 5′ → 3′ direction.

PCR – Direction of Synthesis

  • DNA Polymerase III writes the new strand in the 5′ → 3′ direction.
  • The template strands are read 3′ → 5′.

PCR – Product Length & Yield

  • Typical PCR product length can vary; example calculations show how many base pairs are in the amplified fragment.
  • Example: a product composed of multiple primer-targeted regions might yield products of sizes such as 30 bp, 100 bp, etc.; total length is the sum of fragment lengths.

Gel Electrophoresis – Separation by Size

  • Process: load DNA into wells, apply electric current; DNA migrates toward the positive electrode (red).
  • Separation by fragment size allows visualization of PCR products and estimate of fragment lengths.

DNA Fingerprinting

  • Applications: crime scene investigations, pathogen detection using PCR and gel electrophoresis.
  • Concept: compare DNA fragment lengths from different individuals.

DNA Fingerprinting – Satellites & Microsatellites

  • Satellites: repetitive, non-coding regions between genes (junk region).
  • Microsatellites: short, tandemly repeated DNA sequences within satellites that vary in copy number among individuals; useful for profiling.

DNA Fingerprinting – Example Interpretation

  • Genotypes can be represented by fragment lengths from PCR:
    • Person A: Genotype 7, 7
    • Person B: Genotype 6, 8
  • The length of PCR products correspond to alleles; comparison of fragment lengths across individuals supports identification.