Genetics, DNA Replication & Mutations – Comprehensive Study Notes

Overview of Genetics

• Goal of genetics: understand the entire nucleotide sequence of an organism (micro- to macro-organisms).
  • Enables detection of mutations, tracing inheritance (parents → offspring → grandparents, etc.).
  • Explains how traits are expressed through the structure & function of DNA and RNA.
  • Connects subtle sequence changes to phenotypic effects and disease.

DNA Structure & Functions

• Genetic material: double-stranded DNA (dsDNA); RNA acts as messenger & functional molecule.
• Information is stored in the order of four bases (A, T/U, C, G).
• Complementary base-pairing makes replication semi-conservative (each daughter molecule contains one parental strand).
• Supercoiling: overwinding of the double helix; relieved by topoisomerases.

Enzymes of DNA Replication ("Main Players")

• Helicase
  • Unzips / unwinds the DNA helix by breaking H-bonds.
• Primase
  • Synthesises a short RNA primer (3′-OH provides starting point for DNA polymerase).
• DNA Polymerase III
  • Adds deoxyribonucleotides to the growing strand (5′→3′ direction).
  • Proof-reads with 3′→5′ exonuclease activity to fix mispaired bases.
• DNA Polymerase I
  • Removes RNA primers.
  • Fills resulting gaps & corrects mismatches.
• Ligase
  • Seals remaining "nicks" by catalysing phosphodiester bonds.
• Topoisomerase
  • Generates transient breaks to relax supercoils.
  • Analogy: cutting one strand of a twisted rubber band so it can unwind.
• DNA Gyrase (Topoisomerase II/IV in bacteria)
  • Another supercoil-removing enzyme essential for prokaryotic replication.

DNA Replication Process

• Parent (template) strands direct synthesis of daughter strands; absolutely know which is which in diagrams.
• Replication fork: region where helicase opens the strands.
• Leading strand
  • Continuously synthesised toward the fork.
• Lagging strand
  • Discontinuous synthesis away from the fork → short fragments called Okazaki fragments.
• Each fragment needs its own RNA primer and later ligation.

The Genetic Code & Codons

• Three consecutive bases = codon.
• Each codon codes for one amino acid or a stop signal (degeneracy of the code).
• Total possible codons: 4^3 = 64 (61 sense + 3 stop).

Mutations

Classification by Cause

• Spontaneous mutation: random replication errors (natural background rate).
• Induced mutation: due to physical/chemical mutagens.
  • Example: Ethidium bromide (intercalator, carcinogenic; used to visualise DNA in gels) → requires strict lab controls.

Classification by Effect/Scope

• Point mutation: affects a single base pair.
  • Addition (+1), deletion (−1), or substitution.
  • Sub-types:
  • Silent: codon changes but amino acid remains the same (redundancy of code).
  • Missense: codon change → different amino acid.
  • Nonsense: codon becomes premature stop.
  • Frameshift: insertion/deletion that shifts the reading frame.
• Lethal mutation: disrupts essential functions → cell death.
• Neutral mutation: no significant phenotypic effect.

Frameshift Example (mRNA)

• Original: \text{UUA} \rightarrow \text{Leu}
• Insert G after first U → \text{UGUA} (reading frame shifts right) → downstream codons change.
• Deletion would shift left.

Substitution Examples

• \text{UUA} \rightarrow \text{CUA} (silent; still Leu).
• \text{UUA} \rightarrow \text{GUA} (missense; Val instead of Leu).
• Any mutation producing UAA, UAG or UGA early → nonsense (premature stop).

Repair & Gene Therapy Notes

• Natural repair enzymes (e.g., mismatch repair, excision repair) correct many errors.
• Experimental gene therapy: deliberately edit sequences to restore normal function—promising for HIV/AIDS and other genetic diseases.

Horizontal Gene Transfer (HGT) in Bacteria & Antibiotic Resistance

• Five known HGT mechanisms; three detailed here (others include transduction & transposition).
• Conjugation (Direct)
  • Donor & recipient must touch via a sex pilus.
  • Transfers plasmids or chromosomal segments → may confer:
  • Drug resistance genes.
  • Toxin production genes.
  • Enzymes that metabolise antibiotics.
  • Altered surface proteins (immune evasion).
• Transformation (Indirect)
  • Free DNA fragments/plasmids in environment integrated by competent cells.
  • Laboratory cloning: splice gene of interest into plasmid → introduce via lipofection or electroporation (electric shock opens pores).
• General Notes on Remaining Methods (mentioned but not described)
  • Transduction (phage-mediated DNA transfer).
  • Transposons & integrons (mobile genetic elements).

Practical / Experimental Techniques & Safety

• Electroporation: brief high-voltage pulse creates transient membrane pores.
• Lipid vesicle (liposome) delivery: fuses with membrane to deliver plasmid.
• Mutagen handling (e.g., ethidium bromide): requires controlled, well-labelled, restricted areas to prevent accidental exposure.

Connections, Implications & Real-World Relevance

• Mutation + HGT underlie rapid emergence of multidrug-resistant pathogens.
• Understanding replication enzymes informs antibiotic targets (e.g., quinolones inhibit DNA gyrase).
• Codon usage and silent mutations influence recombinant protein expression strategies.
• Gene-editing therapies rely on precise knowledge of mutation types & repair pathways.