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📚 BIOCHEMISTRY GENETICS — FULL NOTES

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PART 1: DNA STRUCTURE & THE GENOME

1.1 The Double Helix

• DNA is a double-stranded helix composed of deoxyribonucleotides
• Each nucleotide = deoxyribose sugar + phosphate group + nitrogenous base
• Purines: Adenine (A), Guanine (G) — double-ring structures
• Pyrimidines: Cytosine (C), Thymine (T) — single-ring structures
• Base pairing rules (Chargaff's rules): A=T (2 hydrogen bonds), G≡C (3 hydrogen bonds)
• The two strands are antiparallel (one runs 5'→3', the other 3'→5')

1.2 Genome Organisation (Human)

• ~3 billion base pairs (haploid genome)
• ~20,000 protein-coding genes (but genome encodes many hundreds of thousands of proteins via alternative splicing, post-translational modifications)
• Genes cluster in certain chromosome regions; "gene deserts" of >1 Mb exist
• Protein-coding genes + noncoding RNA (ncRNA) genes together regulate phenotype
• Average gene has ~9 exons spanning ~25 kb; introns dominate gene length
• Notable large genes: DMD (dystrophin, >2 Mb on X chromosome), CTNAP2 (>2 Mb on chr 7)

1.3 Gene Structure

1.4 Gene Families & Pseudogenes

• Many genes belong to gene families sharing related sequences (e.g., haemoglobin α- and β-globin clusters on chromosomes 16 and 11)
• Pseudogenes: non-functional copies that arose by gene duplication or retrotransposition

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PART 2: DNA REPLICATION

2.1 Key Principles

• Replication is semiconservative: each daughter molecule retains one parental strand
• Proceeds bidirectionally from origins of replication
• In prokaryotes: single origin (oriC); in eukaryotes: multiple origins (allows replication of large genomes in reasonable time)
• New DNA is only synthesised in the 5'→3' direction; template is read 3'→5'

2.2 Prokaryotic Replication (Key Enzymes)

2.3 Leading vs. Lagging Strand

• Leading strand: synthesised continuously toward the replication fork (5'→3')
• Lagging strand: synthesised discontinuously away from the fork as Okazaki fragments (~1000–2000 nt in prokaryotes; ~100–200 nt in eukaryotes)
• Okazaki fragments are later joined by ligase after RNA primers are replaced by DNA (Pol I in prokaryotes; RNase H + FEN1 in eukaryotes)

2.4 Eukaryotic DNA Polymerases

2.5 The Eukaryotic Cell Cycle

• G1 → S phase (DNA synthesis) → G2 → M (mitosis)
• Cells that stop dividing enter G0 (quiescent)
• Cell-cycle progression is controlled by cyclins and cyclin-dependent kinases (Cdks)
• Checkpoints prevent entry into the next phase until prior phase is complete

2.6 Fidelity of Replication

• DNA Pol errs ~once per 10 million bp; proofreading corrects >99.9% of errors
• Net error rate: ~1 × 10⁻¹⁰ per bp per cell division
• New mutation rate from replication: ~1.2 × 10⁻⁸ per bp per generation (from WGS trio studies)

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PART 3: TRANSCRIPTION

3.1 Overview

• The transfer of genetic information from DNA → RNA
• In eukaryotes, performed by RNA Pol II for mRNA (protein-coding genes); RNA Pol I for rRNA; RNA Pol III for tRNA/5S rRNA
• In bacteria, a single DNA-dependent RNA polymerase performs all transcription; its σ (sigma) factor recognises the promoter
• Rifampin inhibits bacterial RNA polymerase — used in tuberculosis treatment

3.2 Steps of Eukaryotic Transcription

1. Initiation: RNA Pol II binds promoter (with transcription factors); begins at transcriptional start site (TSS = 5' end of mRNA)
2. Elongation: RNA synthesised 5'→3'; template strand read 3'→5'. The coding (sense) strand matches the RNA sequence (with U replacing T)
3. Termination: Transcription extends past the coding region; termination signals are unclear

3.3 RNA Processing (Pre-mRNA → mRNA, in nucleus)

1. 5' capping: 7-methylguanosine cap added — protects mRNA, aids ribosome binding
2. 3' polyadenylation: Cleavage at polyadenylation signal (AAUAAA); poly-A tail added — increases mRNA stability
3. RNA splicing: Introns excised by the spliceosome (snRNPs); exons joined
4. Processed mRNA transported from nucleus → cytoplasm for translation

3.4 Bacterial Gene Regulation (Operon Model)

• Genes grouped in operons (polycistronic); co-regulated
• Example: trp operon — dual control by repressor-co-repressor mechanism AND attenuation
  • High tryptophan → tryptophan binds repressor → active repressor blocks operator → no transcription
  • Attenuation: leader peptide with tandem Trp codons; ribosome stalling forms antiterminator hairpin when Trp is scarce → full transcription proceeds
• Quorum sensing: bacteria regulate virulence genes based on population density (e.g., S. aureus, Pseudomonas)

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PART 4: TRANSLATION & THE GENETIC CODE

4.1 The Genetic Code

• mRNA read in triplets (codons) — 3 bases = 1 codon
• 4³ = 64 possible codons encoding 20 amino acids → the code is degenerate (redundant)
• Wobble position: 3rd codon base tolerates mismatches → multiple codons for same amino acid
• Stop codons: UAA, UAG, UGA (none encode amino acid)
• Start codon: AUG → encodes methionine (fMet in bacteria)
• Methionine and tryptophan are each encoded by a single codon

4.2 Components of Translation

• Ribosome subunits: Prokaryotes = 70S (30S + 50S); Eukaryotes = 80S (40S + 60S)
• rRNA genes: 18S and 28S in eukaryotes

4.3 Stages of Translation

1. Initiation: Ribosome assembles at start AUG; initiator tRNA (fMet-tRNA in bacteria) binds P-site
2. Elongation: Aminoacyl-tRNA enters A-site → peptide bond forms → ribosome translocates one codon → tRNA exits via E-site; cycle repeats
3. Termination: Stop codon in A-site → release factors bind → polypeptide released → ribosome dissociates

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PART 5: MUTATIONS

5.1 Types of Mutations

Point Mutations (single base changes)

Frameshift Mutations

• Insertion or deletion of bases (not a multiple of 3) → shifts reading frame → completely different amino acid sequence downstream → usually hits a premature stop
• Insertion or deletion of 1–2 bp causes frameshifts; insertions/deletions of 3 bp (in-frame) do not frameshift

Large-Scale Mutations

5.2 Causes of Mutation

• DNA replication errors (corrected by proofreading; residual error ~10⁻¹⁰/bp)
• Spontaneous deamination of 5-methylcytosine → Thymine at CpG dinucleotides — most common spontaneous mutation, >30% of all SNVs; occurs 25× more frequently than other SNVs → CpG is a mutational hot spot
• Depurination, depyrimidination, oxidative damage

• UV light → pyrimidine dimers (T-T dimers); repaired by nucleotide excision repair (NER)
• Alkylating agents, intercalating agents, ionising radiation

5.3 DNA Repair Mechanisms

• Defective NER → Xeroderma pigmentosum (extreme UV sensitivity, skin cancer)
• Defective MMR → Lynch syndrome (hereditary colorectal cancer)
• Defective BRCA1/2 (HR) → breast/ovarian cancer

5.4 Mutation Rate Data

• Overall rate per genome per generation: ~1.2 × 10⁻⁸ per bp
• Median gene mutation rate: ~1 × 10⁻⁶ per locus per generation
• Example: Achondroplasia — new mutation rate ~1.4 × 10⁻⁵/locus/generation; virtually all cases caused by the same G→A transition (Gly→Arg) at a CpG hot spot in FGFR3

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PART 6: PATTERNS OF INHERITANCE

6.1 Mendelian Principles

• Segregation: alleles separate during gamete formation; offspring receive one allele per locus
• Independent Assortment: alleles at different loci assort independently (unless linked)
• Dominance: one allele may mask the other

6.2 Autosomal Recessive (AR) Inheritance

• Disease occurs only in homozygotes (aa) or compound heterozygotes (a¹/a²) — no functioning allele
• Both parents are typically carriers (Aa): each pass pathogenic allele 50% of the time
• Risk: 25% affected, 50% carrier, 25% unaffected (from two carrier parents)
• Usually loss-of-function mutations (enzyme deficiencies)
• Affected individuals often in same sibship; parents unaffected
• Sex-influenced AR: e.g., hereditary haemochromatosis — more common/severe in males (iron not lost via menstruation)

6.3 Autosomal Dominant (AD) Inheritance

• Disease occurs in heterozygotes (Aa) — one pathogenic allele sufficient
• Risk: 50% for each offspring of affected parent
• Often gain-of-function mutations or haploinsufficiency
• Vertical transmission (affected in each generation)
• New mutations common (e.g., achondroplasia — FGFR3 gain-of-function)

6.4 X-Linked Inheritance

• Gene on the X chromosome
• X-linked recessive: males (hemizygous, XY) affected; females are carriers
  • Affected sons from carrier mothers; no father-to-son transmission
  • Examples: haemophilia A/B, Duchenne muscular dystrophy (DMD), G6PD deficiency
• X-linked dominant: heterozygous females affected; males often more severely affected
  • Example: Fragile X syndrome, Rett syndrome (MECP2)

6.5 ABO Blood Group (Codominance)

• A and B alleles are codominant; O is recessive to both A and B

6.6 Chromosomal Abnormalities

• Nondisjunction during meiosis → trisomy/monosomy
  • Meiotic nondisjunction (especially in oogenesis) = most common mutational mechanism in humans
  • Leads to chromosomally abnormal fetuses in several % of all pregnancies
• Trisomy 21 (Down syndrome): extra chromosome 21; most common viable trisomy
• Turner syndrome (45,X): monosomy X; short stature, ovarian dysgenesis
• Klinefelter syndrome (47,XXY)
• Mosaic forms arise from mitotic nondisjunction after fertilisation

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PART 7: GENE EXPRESSION REGULATION

7.1 Levels of Regulation

1. Chromatin remodelling (histones, nucleosome positioning)
2. DNA methylation (epigenetic — 5-methylcytosine at CpG; generally suppresses gene expression)
3. Transcriptional control (transcription factors, enhancers, promoters)
4. Post-transcriptional (alternative splicing, RNA stability)
5. Translational control (miRNA, siRNA, ribosome activity)
6. Post-translational (phosphorylation, ubiquitination, glycosylation)

7.2 Alternative Splicing

• The same pre-mRNA can be spliced differently → multiple proteins from one gene
• ~20,000 protein-coding genes → hundreds of thousands of proteins via alternative splicing
• Tissue-specific and developmentally regulated

7.3 Non-Coding RNAs

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PART 8: MOLECULAR TECHNIQUES

8.1 Polymerase Chain Reaction (PCR)

• Amplifies specific DNA sequences in vitro
• Components: Template DNA, primers (2 oligonucleotides flanking target), dNTPs, Taq polymerase, buffer
• Cycle (repeated ~30×): Denaturation (94°C) → Annealing (55–65°C) → Extension (72°C)
• Result: exponential amplification (~2ⁿ copies after n cycles)
• Variants: RT-PCR (from mRNA), qPCR (quantitative/real-time), allele-specific PCR

8.2 Gel Electrophoresis

• Separates DNA/RNA/proteins by size and charge through a gel matrix (agarose for nucleic acids; polyacrylamide for proteins)
• DNA migrates toward positive electrode (negatively charged at neutral pH)
• Ethidium bromide (or SYBR Green) staining visualises bands under UV light
• Smaller fragments migrate further

8.3 Southern Blotting

• Detects specific DNA sequences in a genome
• Steps: Digest DNA with restriction enzymes → gel electrophoresis → transfer to membrane → hybridise with labelled probe → detect
• Used for: gene copy number, RFLP analysis, diagnosing large deletions

8.4 Northern Blotting

• Same principle as Southern but detects RNA (mRNA expression levels)

8.5 DNA Sequencing

• Sanger sequencing (chain termination): uses dideoxynucleotides (ddNTPs) to terminate elongation at specific bases; fragments separated by size → sequence read
• Next-Generation Sequencing (NGS): massively parallel sequencing; millions of reads simultaneously; enables Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES)

8.6 Restriction Enzymes

• Bacterial endonucleases that cut DNA at specific palindromic sequences
• Produce blunt ends or sticky ends (overhangs)
• Essential tools for cloning, restriction mapping, and Southern blotting

8.7 Recombinant DNA & Cloning

• Cut both gene of interest and vector (plasmid/viral) with same restriction enzyme → mix and ligate → transform into host bacteria → select colonies with insert
• Applications: protein production (insulin, growth hormone), gene therapy vectors, research tools

8.8 FISH (Fluorescence In Situ Hybridisation)

• Fluorescently labelled DNA probes hybridise to specific chromosomal loci
• Detects chromosomal deletions, duplications, translocations in intact cells
• Used in diagnosis of microdeletion syndromes (DiGeorge/22q11.2), cancer cytogenetics

8.9 Array CGH / SNP Arrays

• Genome-wide detection of copy number variants (CNVs) and loss of heterozygosity
• Patient DNA co-hybridised with reference DNA to microarray → relative fluorescence ratios reveal gains/losses

8.10 CRISPR-Cas9 Gene Editing

• Guide RNA (gRNA) directs Cas9 endonuclease to a specific genomic sequence
• Cas9 creates a double-strand break → repaired by NHEJ (introduces mutations) or HDR (precise edits)
• Applications: disease modelling, gene therapy, functional genomics

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QUICK REFERENCE: Key Numbers to Know

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QUICK REFERENCE: Important Clinical Connections

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