Molecular Biology & Genetics Fundamentals

MolBio integrates biochem, genetics, and cell bio to explain how living systems store, transmit, and express genetic information at the molecular level.

## DNA Structure & Replication

DNA double helix: antiparallel strands connected by H-bonds between complementary bases — A=T (2 H-bonds), G≡C (3 H-bonds). Sugar-phosphate backbone w/ 3'→5' phosphodiester bonds. B-form DNA: 10 bp per turn, 3.4 nm pitch, 2 nm diameter. Major groove (protein binding, TF recognition) and minor groove.

Replication is semiconservative (Meselson-Stahl experiment). Origin of replication (ORI): prokaryotes have 1 (oriC), eukaryotes have many. Helicase unwinds, SSB proteins stabilize, topoisomerase relieves torsional strain. Primase synthesizes RNA primer. DNA Pol III (prokaryotes) or Pol δ/ε (eukaryotes) extends 5'→3' only.

Leading strand: continuous synthesis toward replication fork. Lagging strand: discontinuous Okazaki fragments (100-200 nt in eukaryotes, 1000-2000 in prokaryotes). DNA Pol I removes RNA primers and fills gaps. DNA ligase seals nicks. Proofreading: 3'→5' exonuclease activity of DNA Pol, error rate ~10⁻⁷ per bp after MMR.

Telomeres: TTAGGG repeats (human) at chromosome ends, shortened each division (end replication problem). Telomerase (reverse transcriptase + RNA template) extends telomeres — active in stem cells and cancer cells, silenced in most somatic cells. Telomere shortening → replicative senescence → Hayflick limit (~50 divisions).

## Gene Expression: Transcription

Prokaryotic: RNA Pol (single enzyme, 5 subunits) binds promoter (σ factor recognizes -10 Pribnow box TATAAT and -35 region). Transcription and translation coupled (no nuclear envelope). Operons: lac operon — negative control (repressor binds operator when no lactose) + positive control (CAP-cAMP when glucose low). Polycistronic mRNA.

Eukaryotic: three RNA Pols — Pol I (rRNA), Pol II (mRNA, snRNA), Pol III (tRNA, 5S rRNA). Promoter elements: TATA box (~-25), Inr. General TFs assemble at promoter: TFIID (TBP subunit binds TATA) → TFIIA,B → Pol II → TFIIF,E,H. Enhancers can be thousands of bp away, act through DNA looping + Mediator complex.

mRNA processing (eukaryotes only): 5' cap (7-methylguanosine, added co-transcriptionally, protects from degradation, aids ribosome binding), 3' poly-A tail (50-250 adenines, added by PAP after cleavage at AAUAAA signal, increases stability and export). Splicing: introns removed by spliceosome (snRNPs U1,U2,U4,U5,U6). Lariat intermediate. Alternative splicing → multiple proteins from one gene (Drosophila DSCAM: 38,000+ variants).

## Gene Expression: Translation

Genetic code: 64 codons, 61 sense + 3 stop (UAA, UAG, UGA). Degenerate (multiple codons per AA) but unambiguous (each codon = one AA). Start codon AUG (Met). Wobble position: 3rd base of codon allows non-Watson-Crick pairing → fewer tRNAs needed than 61.

tRNA: cloverleaf secondary structure, L-shaped 3D. Aminoacyl-tRNA synthetases charge tRNAs (highly specific, one per AA). Proofreading via editing site ensures correct AA-tRNA pairing. Anticodon pairs w/ mRNA codon (antiparallel).

Ribosome: 70S prokaryotic (30S + 50S), 80S eukaryotic (40S + 60S). Three sites: A (aminoacyl/incoming), P (peptidyl/growing chain), E (exit). Initiation: prokaryotes — Shine-Dalgarno sequence on mRNA recruits 30S. Eukaryotes — 5' cap scanning by 40S + eIF4E until first AUG (Kozak context).

Elongation: EF-Tu delivers aminoacyl-tRNA to A site (GTP hydrolysis for proofreading), peptide bond formation (peptidyl transferase = rRNA, ribozyme), translocation by EF-G moves ribosome one codon 5'→3'. ~15-20 AA/sec in prokaryotes. Polyribosomes: multiple ribosomes translate same mRNA simultaneously.

Termination: release factors (RF1/RF2 prokaryotes, eRF1 eukaryotes) recognize stop codons, catalyze peptide release. Post-translational modifications: folding (chaperones HSP60, HSP70), cleavage (signal peptide removal), glycosylation (ER/Golgi), phosphorylation (kinases), ubiquitination (proteasome targeting).

## Genetics & Inheritance

Mendelian: Law of Segregation (alleles separate in meiosis I). Law of Independent Assortment (genes on different chromosomes sort independently). Dominance: complete, incomplete (intermediate phenotype), codominance (both expressed, e.g., AB blood type).

Chromosome theory: genes on chromosomes, linked genes deviate from independent assortment. Recombination frequency = (recombinant offspring/total) × 100. 1 map unit (cM) = 1% recombination. Max measurable distance ~50 cM (appears unlinked beyond this). Three-point cross to determine gene order and map distances.

Sex-linked: X-linked recessive (hemophilia, color blindness) — affected males hemizygous, carrier females heterozygous. X-inactivation: one X randomly silenced in each cell (Barr body), females are mosaics (calico cats). Y-linked: few genes (SRY = male determination).

Epigenetics: heritable changes w/o DNA sequence alteration. DNA methylation (CpG islands, typically silencing). Histone modifications: acetylation (HAT → open chromatin, active), deacetylation (HDAC → closed, silent), methylation (context-dependent). Genomic imprinting: parent-of-origin expression (Igf2 paternal, H19 maternal).

## Molecular Techniques

PCR: denature (94-98°C) → anneal primers (50-65°C) → extend (72°C, Taq polymerase). 2ⁿ amplification after n cycles. Primer design: 18-25 nt, 40-60% GC, Tm matched within 5°C, avoid self-complementarity. RT-PCR for RNA → cDNA first. qPCR (real-time): Ct value inversely proportional to initial template amount.

Gel electrophoresis: agarose (DNA, 0.5-2%) or PAGE (proteins). DNA migrates toward + electrode (neg charged phosphate backbone), smaller fragments migrate faster. EtBr or SYBR staining for visualization. SDS-PAGE: SDS denatures and coats proteins uniformly → separation by MW only. Western blot: SDS-PAGE → transfer to membrane → antibody detection.

CRISPR-Cas9: guide RNA (gRNA, 20 nt) directs Cas9 nuclease to complementary DNA sequence adjacent to PAM (NGG for SpCas9). Creates DSB → repaired by NHEJ (insertions/deletions, gene knockout) or HDR (precise editing w/ donor template, less efficient). Off-target effects: gRNA may bind similar sequences elsewhere. Base editors and prime editors for single-base changes w/o DSB.

Cloning: restriction enzymes cut at palindromic sequences (EcoRI: GAATTC → sticky ends). Ligate insert into vector (plasmid, BAC, viral) w/ DNA ligase. Transform into E. coli (heat shock or electroporation). Screen: blue-white (lacZ disruption), antibiotic resistance, colony PCR. Expression vectors include promoter, RBS, MCS, terminator, selection marker.

Sequencing: Sanger (chain termination, ddNTPs, gold standard for single genes, read length ~800 bp). NGS: Illumina (short reads 150-300 bp, high throughput, sequencing by synthesis), Oxford Nanopore (long reads >100 kb, real-time, portable MinION). Applications: WGS, RNA-seq (transcriptome), ChIP-seq (protein-DNA interactions), ATAC-seq (chromatin accessibility).