DNA and Biotechnology

6.1 DNA Structure

This section covers the basic structure of DNA and RNA, including nucleosides, nucleotides, sugar-phosphate backbone, purines/pyrimidines, Watson-Crick model, DNA denaturation, and reannealing.

• Nucleosides and Nucleotides
  • Nucleosides = pentose sugar + nitrogenous base (linked to C1').
  • Nucleotides = nucleoside + phosphate group (attached to C5').
  • RNA: ribose (2' OH); DNA: deoxyribose (2' H).
  • Example: Adenine → adenosine; AMP = nucleotide.
• Sugar-Phosphate Backbone
  • Alternating sugar & phosphate groups.
  • Directionality: 5' → 3'.
  • Nucleotides joined by 3'→5' phosphodiester bonds.
  • 5' end: OH or phosphate on C5'.
  • 3' end: free OH on C3'.
  • dsDNA = double stranded; sRNA = single stranded.
• Purines and Pyrimidines
  • Purines: 2 rings (A & G).
  • Pyrimidines: 1 ring (C, T, U).
  • Aromatic, cyclic, planar, conjugated (Hückel's rule: 4n+2 π e⁻).
  • N atoms confer stability.

  

• Watson and Crick Model
  • 1953: double helix, antiparallel, complementary base pairing.
  • Chargaff’s rules: A=T, C=G.
  • B-DNA: right-handed, 3.4 nm turn, 10 bp per turn; major/minor grooves.
  • Z-DNA: left-handed, zigzag, 12 bp per 4.6 nm; high GC/salt.

  

• Denaturation and Reannealing
  • Denatured by heat, high pH, chemicals.
  • Reannealed if denaturant removed.
  • Important for PCR; probe DNA binds target sequences.

6.2 Eukaryotic Chromosome Organization

Describes the packaging of eukaryotic DNA, histones, nucleosomes, chromatin types, telomeres, and centromeres.

• Histones
  • DNA wrapped around basic histone proteins → chromatin.
  • 5 histone types: H2A, H2B, H3, H4 (2 copies → histone core), H1.
  • 200 bp wrapped → nucleosome ("beads on a string").
  • Histones = nucleoproteins.

  

• Heterochromatin vs Euchromatin
  • Heterochromatin: compact, dark, transcriptionally silent.
  • Euchromatin: dispersed, light, active transcription.
• Telomeres
  • Repeating sequence: TTAGGG at DNA ends.
  • Prevent loss of info; replaced by telomerase.
  • High GC content → stable.
  • Progressive shortening → aging

  

• Centromeres
  • DNA region at chromosome center; site of constriction.
  • Composed of heterochromatin; high GC tandem repeats.
  • Sister chromatids remain connected until microtubules separate.

6.3 DNA Replication

Covers the mechanisms of DNA replication including origin of replication, helicase activity, DNA polymerases, leading/lagging strands, Okazaki fragments, and replication of chromosome ends.

• Strand Separation
  • Origins of replication: unwinding DNA → replication forks.
  • Bacteria: circular, single origin → 2 forks.
  • Eukaryotes: linear, multiple origins → sister chromatids.
• Helicase & Topoisomerase
  • Helicase unwinds DNA → single-stranded templates.
  • Positive supercoiling occurs → topoisomerase introduces negative supercoils.
  • Single-strand binding proteins prevent reassociation/degradation.
• Parent & Daughter Strands
  • Semiconservative replication.
  • DNA pol reads 3'→5', synthesizes 5'→3'.
  • Leading strand: continuous replication.
  • Lagging strand: discontinuous → Okazaki fragments.
• Replication Steps
  • Primase lays RNA primer.
  • DNA pol III/α/δ synthesize daughter strands.
  • DNA pol I/RNase H remove RNA primer.
  • DNA pol I/δ fill in nucleotides.
  • DNA ligase seals fragments.
• Eukaryotic DNA pol
  • α, δ, ε: nuclear replication.
  • γ: mitochondrial DNA.
  • β, ε: DNA repair.
  • δ & ε assisted by PCNA (sliding clamp).
• Replicating Chromosome Ends
  • DNA pol cannot complete 5' ends → telomere shortening.

6.4 DNA Repair

Discusses types of DNA damage, oncogenes/tumor suppressors, and mechanisms of repair including proofreading, mismatch repair, nucleotide/base excision repair.

• DNA Damage
• Causes: chemicals, radiation, backbone break, base alterations, replication errors → ↑ cancer risk.
• Oncogenes & Tumor Suppressors
  • Oncogenes: mutated proto-oncogenes → dominant, promote proliferation.
  • Tumor suppressors: inhibit cell cycle (e.g., p53, Rb); loss → cancer.
• Proofreading & Mismatch Repair
  • DNA pol proofreading: detects unstable H bonds; template strand methylated.
  • Mutations higher in lagging strand.
  • Mismatch repair: G2 machinery (MSH2, MLH1) fixes replication errors missed in S phase.
• Nucleotide & Base Excision Repair
  • Nucleotide: UV-induced thymine dimers → excision endonuclease, DNA pol, ligase.
  • Base: small base modifications (e.g., C→U) → glycosylase removes base → AP site → AP endonuclease → DNA pol & ligase fill/repair.

6.5 Recombinant DNA and Biotech

Explains recombinant DNA technology, cloning, restriction enzymes, DNA libraries, PCR, gel electrophoresis, DNA sequencing, gene therapy, transgenic mice, and associated ethical concerns.

• Recombinant DNA Technology
  • Multiply DNA fragments via cloning or PCR.
  • Alters genes/proteins; source for recombinant proteins (e.g., insulin).
• DNA Cloning & Restriction Enzymes
  • DNA ligated into vector → recombinant vector.
  • Vectors: bacterial/viral plasmids → transferred to host → colonies → express gene.
  • Restriction enzymes: recognize specific sequences; can create sticky ends.
• DNA Libraries
  • Genomic: coding + noncoding DNA fragments.
  • cDNA: reverse transcribed from RNA → only expressed sequences (expression library).
• Hybridization
  • Complementary base pairing using single-stranded sequences.
• PCR
  • Amplifies DNA millions of times; requires primers, heat, reannealing.
  • High GC content primers → stable.
• Southern Blot & Gel Electrophoresis
  • Agarose gel separates DNA by size/charge.
  • Southern blot: cut fragments → membrane → labeled probe binds complementary DNA.
• DNA Sequencing
  • Dideoxy nucleotides (ddATP, ddCTP, etc.) terminate chain.
  • Fragments separated by size → sequence determined.
• Applications
  • Gene Therapy: normal gene introduced to correct mutation; virus vectors common; risk of oncogene activation.
  • Transgenic Mice: cloned gene (transgene) microinjected into fertilized ova or stem cells; passed to offspring.
    • Chimera formation if stem cells mixed with blastocytes.
    • Knockout mice: gene deletion → disease study.
• Safety & Ethics
  • Risks: pregnancy termination, privacy concerns, consent for subjects unable to communicate.

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