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1. Regulation of Gene Expression

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Prokaryotic Regulation - The LAC Operon Model (Jacob & Monod, 1961)

Lac Operon Components:

Two States:

Repressor → binds Operator → blocks RNA Polymerase → NO transcription

Allolactose (inducer) → binds Repressor → Repressor leaves Operator → RNA Polymerase proceeds → Polycistronic mRNA produced → 3 enzymes synthesized

Types of Control:

• Negative control: Repressor blocks transcription (as above)
• Positive control: CAP-cAMP complex activates transcription (when glucose is absent)
• Constitutive expression: Housekeeping genes - always expressed at constant rate

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Eukaryotic Regulation

Key difference: Eukaryotes do NOT have operons. Each gene has its own promoter.

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2. Principle of Electrophoresis

Basic Principle:

Charged molecules placed in an electric field migrate toward the opposite pole.

(−) Cathode  ←—[gel/medium]—→  Anode (+)
              ↑
         Sample loaded here
         (DNA/RNA are -ve charged,
          migrate TOWARD +ve pole)

Factors Governing Migration:

Types of Electrophoresis:

• Agarose gel electrophoresis - separates nucleic acids (DNA, RNA)
• Polyacrylamide gel electrophoresis (PAGE) - separates proteins
• SDS-PAGE - sodium dodecyl sulphate denatures proteins; separation purely by size
• Capillary electrophoresis (CE) - separates proteins in a capillary tube
• Immunofixation electrophoresis - detects monoclonal proteins (M-band in myeloma)

Visualization:

• DNA/RNA - Ethidium bromide stain (fluoresces under UV)
• Proteins - Coomassie blue, Amido black, Ponceau S stain

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3. Northern Blotting

Named "Northern" as a directional analogy to "Southern" blotting (which uses DNA).

Steps (Mnemonic: ETHER-H)

Simple Flow Diagram:

Tissue Sample
     ↓
Extract mRNA
     ↓
Run on Agarose-Formaldehyde Gel
     ↓
Transfer mRNA to Nitrocellulose Membrane
     ↓
Hybridize with Labeled DNA Probe
     ↓
Wash (remove unbound probe)
     ↓
Autoradiography / Fluorescence Detection
     ↓
BAND = Size and Amount of specific mRNA

What Northern Blot Tells Us:

Comparison of Blotting Techniques:

Clinical Significance:

• Detects abnormal mRNA in genetic diseases
• Studies gene expression in cancer vs normal tissue
• Confirms results of PCR or microarray experiments
• Now largely replaced by RT-PCR and RNA sequencing (but still the only method giving mRNA size information)

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Sources: Harper's Illustrated Biochemistry 32nd Ed; Basic Medical Biochemistry - A Clinical Approach 6e; Emery's Medical Genetics; Schwartz's Principles of Surgery 11th Ed

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1. Chromatography - Principle & Types

Principle

Chromatography is a separation technique based on the differential distribution of compounds between a mobile phase and a stationary phase as they travel through a support medium.

• Compounds that interact more strongly with the stationary phase are retained longer (elute later)
• Compounds that interact more with the mobile phase move faster (elute earlier)
• Each compound has a characteristic retention time (tR) - used to identify it

Key Terms:

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Types of Chromatography

1. Ion Exchange Chromatography

• Separates molecules based on charge
• Cation exchange resin (negatively charged) - binds positively charged molecules
• Anion exchange resin (positively charged) - binds negatively charged molecules
• Elution done by changing salt concentration or pH
• Use: Purification of proteins, amino acids, nucleotides

2. Gel Filtration / Size Exclusion Chromatography

• Separates molecules based on size (molecular weight)
• Column packed with porous beads
• Large molecules - cannot enter pores → elute FIRST
• Small molecules - enter pores → elute LAST
• Use: Determining molecular weight, protein purification

Large molecule → excluded from pores → elutes FIRST
Small molecule → enters pores → elutes LAST

3. Affinity Chromatography

• Most specific type of chromatography
• Column has a specific ligand (e.g., antibody, enzyme substrate) covalently attached
• Only the target molecule binds; others pass through
• Target eluted by salt gradient or specific substrate
• Use: Purification of antibodies, enzymes, receptors, hormones

4. Adsorption / Partition Chromatography

• Paper Chromatography: Stationary = water in paper; Mobile = organic solvent
• TLC (Thin Layer): Stationary = silica on plate; Mobile = solvent
• Separation by polarity - polar compounds stay near origin; nonpolar move far
• Use: Amino acid analysis (ninhydrin spray), lipid separation

5. Gas Chromatography (GC)

• Mobile phase = inert gas (He, N₂)
• Stationary phase = liquid on column packing
• Requires volatile compounds; temperature-controlled oven
• Use: Drug detection, toxicology, amino acid analysis, fatty acid profiling

6. High-Performance Liquid Chromatography (HPLC)

• Mobile phase = liquid pumped under high pressure
• Very high resolution, sensitivity, speed
• Use: Drug levels (immunosuppressants, antibiotics), hormone assays, HbA1c

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2. Products Derived from Glycine

Summary Table:

Memory Aid - "HCGBPSO":

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3. Salient Features of the Genetic Code

• 4 bases → 3-base codons → 4³ = 64 possible codons for 20 amino acids
• First cracked by Marshall Nirenberg (1961) - poly(U) → polyphenylalanine (UUU = Phe)

The 8 Salient Features (Mnemonic: "T-DUNDCN"):

Codon Chart Summary:

64 total codons:
  ├── 61 → amino acids (sense codons)
  │     ├── AUG → Methionine (START, only codon for Met)
  │     └── UGG → Tryptophan (only codon for Trp)
  └──  3 → STOP codons (UAA, UAG, UGA)
        (Mnemonic: "U Are Away", "U Are Gone", "U Go Away")

Mutations and Consequences:

• Silent mutation - codon changed but same amino acid (e.g., UCA → UCU = still Ser)
• Missense mutation - different amino acid coded (e.g., Sickle cell: GAG → GUG = Glu → Val)
• Nonsense mutation - premature stop codon → truncated protein
• Frameshift - insertion/deletion shifts reading frame → completely different protein downstream

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4. Quality Control (QC) in Biochemistry Laboratory

Three Phases of Quality Control:

A. Pre-analytical QC

• Patient preparation (fasting, medication effects)
• Correct sample collection (right tube, anticoagulant)
• Proper labeling
• Transport and storage conditions (temperature, time)
• Hemolysis, lipemia, icterus - pre-analytical interferences

B. Analytical QC (most important for exam)

• Normal control - values in normal range
• Abnormal control - values in high/low range

+3 SD  ----warning----
+2 SD  ----warning----
+1 SD  
Mean   ═══════════════
-1 SD  
-2 SD  ----warning----
-3 SD  ----warning----

• Random error - unpredictable, affects precision (e.g., air bubble in pipette)
• Systematic error - consistent bias, affects accuracy (e.g., wrong calibrator, reagent deterioration)

C. Post-analytical QC

• Result interpretation with proper reference ranges
• Reporting critical (panic) values immediately
• Delta check - compare with patient's previous result to detect specimen mix-up
• Proper transcription and reporting

Internal vs External QC:

Key Exam Fact: Accuracy is assessed by bias from true value; Precision is assessed by CV% (coefficient of variation) = (SD/mean) × 100. A good lab has CV% < 5% for most assays.

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Sources: Henry's Clinical Diagnosis & Management by Laboratory Methods; Tietz Textbook of Laboratory Medicine 7th Ed; Lippincott Illustrated Reviews Biochemistry 8th Ed; Basic Medical Biochemistry - A Clinical Approach 6e

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1. Tumour Markers (25 Important Markers)

Key Point: No tumour marker is 100% specific or 100% sensitive. They are NEVER used alone for diagnosis.

Complete Table of 25 Important Tumour Markers:

Uses of Tumour Markers:

1. Screening - population at high risk (e.g., AFP in cirrhosis for HCC)
2. Diagnosis - support, never sole basis
3. Staging - gauge extent of disease
4. Monitoring treatment - falling levels = good response
5. Detecting recurrence - rising levels post-treatment = relapse
6. Prognosis - e.g., LDH in lymphoma staging

Oncofoetal Antigens (important exam category):

• AFP - produced by fetal liver / yolk sac; re-expressed in HCC
• CEA - produced during fetal development; re-expressed in GI cancers

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2. Gene Therapy

Types:

Requirements Before Gene Therapy Can Be Applied (Box 14.1):

1. Identity of the molecular defect must be known
2. A functional cDNA/gene clone must be available
3. An appropriate vector must be available
4. A target cell accessible for gene delivery must exist

Methods of Gene Delivery (Vectors):

A. Viral Vectors (most efficient):

RETROVIRUS
  → Integrates into host genome
  → Dividing cells only
  → Used for: SCID (X-linked), ADA-SCID
  → Risk: insertional mutagenesis

ADENOVIRUS
  → Does NOT integrate (episomal)
  → Infects non-dividing cells too
  → Used for: Cystic fibrosis, cancer (transient effect)
  → Risk: immune reaction

ADENO-ASSOCIATED VIRUS (AAV)
  → Episomal (stable without integration)
  → Long-term expression in non-dividing cells
  → Used for: Hemophilia B, SMA (Zolgensma), Leber amaurosis
  → FDA APPROVED products available

LENTIVIRUS
  → Integrates into genome
  → Infects non-dividing cells
  → Used for: X-linked adrenoleukodystrophy, SCID (SIN vectors)

B. Non-viral Vectors (less efficient, safer):

• Liposomes - lipid vesicles carry DNA into cell
• Naked DNA injection - direct injection into muscle
• Electroporation - electric pulses open cell membrane pores
• Gene gun - biolistic particle delivery

Examples of Approved/Successful Gene Therapy:

Limitations:

• Insertional mutagenesis - viral integration can activate oncogenes (5 SCID patients got leukemia-like disorder)
• Immune response to viral vector
• Transient expression (for non-integrating vectors)
• Large genes cannot fit in current vectors
• High cost (Zolgensma = ~$2 million per dose)

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3. Quaternary Structure of Protein

Key Points:

• Occurs when a protein has more than one polypeptide chain
• Each polypeptide chain = a subunit (monomer)
• Subunits may be identical (homo-multimer) or different (hetero-multimer)
• Subunits held together by non-covalent bonds primarily

Bonds Holding Subunits Together:

Classic Example - Hemoglobin:

Hemoglobin = TETRAMER
    ┌──────────────────────────────┐
    │   α₁ subunit + heme group   │
    │   α₂ subunit + heme group   │
    │   β₁ subunit + heme group   │
    │   β₂ subunit + heme group   │
    └──────────────────────────────┘
    Total = 4 subunits (2α + 2β)

Other Examples:

Isoenzymes:

• Proteins with same function but different primary structure
• Arise from different genes OR tissue-specific processing
• Have different subunit compositions
• Example: LDH₁ (HHHH - heart) vs LDH₅ (MMMM - liver/muscle)

Significance:

• Quaternary structure allows cooperative interactions
• Enables allosteric regulation
• Provides structural stability
• Isoenzyme patterns used as diagnostic markers (CK-MB = heart; LDH₁ > LDH₂ = MI)

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4. Free Radicals

Types of Reactive Oxygen Species (ROS):

Generation of Free Radicals:

Sources of Free Radicals:

1. Normal metabolism
   O₂ → (4 electrons) → H₂O (normal respiration)
   Partial reduction → O₂•⁻, H₂O₂, •OH (byproducts)

2. Fenton Reaction (Iron catalyzed):
   H₂O₂ + Fe²⁺ → Fe³⁺ + •OH + OH⁻

3. Radiation / UV light:
   H₂O → •OH + H•

4. Leukocyte activation (NADPH oxidase):
   Burst → O₂•⁻ (kills microbes)

5. Drugs/Chemicals:
   CCl₄ → •CCl₃ (liver toxin)

Removal (Defense Mechanisms):

FREE RADICAL REMOVAL SYSTEMS:

1. SOD (Superoxide dismutase)
   2O₂•⁻ + 2H⁺ → H₂O₂ + O₂
   (Mn-SOD = mitochondria; Cu/Zn-SOD = cytoplasm)

2. Catalase (in peroxisomes)
   2H₂O₂ → O₂ + 2H₂O

3. Glutathione Peroxidase
   H₂O₂ + 2GSH → GSSG + 2H₂O

4. Antioxidants (non-enzymatic):
   → Vitamin E (fat-soluble, membrane protector)
   → Vitamin C (ascorbic acid, water-soluble)
   → Vitamin A / Beta-carotene
   → Glutathione (GSH) in cytosol

5. Metal binding proteins:
   → Transferrin, Ferritin (iron) - prevent Fenton reaction
   → Ceruloplasmin (copper)

Pathological Effects (Three Main Reactions):

Clinical Significance:

Memory Aid for ROS scavengers: "SOD-CAT-GLUT" → SOD → Catalase → Glutathione peroxidase

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Sources: Robbins & Kumar Basic Pathology (Robbins Pathology); Robbins, Cotran & Kumar Pathologic Basis of Disease; Lippincott Illustrated Reviews Biochemistry 8th Ed; Thompson & Thompson Genetics & Genomics in Medicine 9th Ed; Bailey & Love's Short Practice of Surgery 28th Ed

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1. Toxins - Causes & Deficiency

Note: In Vasudevan-based biochemistry exams, "Toxins" as a topic is typically asked in the context of vitamin/nutrient deficiency caused by toxins, metabolic toxins (ammonia, bilirubin, homocysteine), and drug/chemical toxins and their biochemical effects.

A. Vitamin Deficiency Caused by Toxins / Drugs

B. Metabolic Toxins (Endogenous):

C. Classic Enzyme Inhibitions by Toxins:

TOXIN              →  ENZYME INHIBITED      →  EFFECT
─────────────────────────────────────────────────────────
Cyanide (CN⁻)     →  Cytochrome c oxidase  →  No ATP (histotoxic hypoxia)
Fluoride (F⁻)     →  Enolase               →  No glycolysis
Lead (Pb²⁺)       →  ALA dehydratase       →  No heme → anemia
Malonate           →  Succinate dehydrogenase →  TCA blocked
Arsenic           →  Pyruvate dehydrogenase →  No acetyl CoA
Mercury           →  SH-group enzymes       →  Widespread enzyme inhibition

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2. miRNA & ncRNA

miRNA (microRNA) - Biogenesis Pathway:

NUCLEUS
  │
  ▼
Gene → transcribed → pri-miRNA (primary miRNA, long hairpin transcript)
  │
  ▼ Microprocessor complex (Drosha + DGCR8)
  │
pre-miRNA (precursor, ~70 nt stem-loop structure)
  │
  ▼ Exportin-5 exports to CYTOPLASM
  │
  ▼ DICER enzyme cleaves pre-miRNA
  │
Mature miRNA duplex (~21-23 nucleotides, single-stranded)
  │
  ▼ Loaded into RISC (RNA-Induced Silencing Complex)
  │
miRNA-RISC binds complementary 3'UTR of target mRNA
  │
  ├── Perfect complementarity → mRNA CLEAVAGE/DEGRADATION
  └── Imperfect complementarity → TRANSLATIONAL REPRESSION
  │
  ▼
GENE SILENCED (protein not produced)

Key Features of miRNA:

• Size: 21-23 nucleotides (single-stranded)
• Location: Encoded in genome; found in nucleus and cytoplasm
• Function: Post-transcriptional gene silencing - one miRNA can regulate hundreds of target mRNAs
• Targets: 3'UTR (untranslated region) of mRNA
• Clinical role: Dysregulated in cancer, cardiac disease; potential biomarkers and therapeutic targets

Types of Non-Coding RNA (ncRNA):

siRNA vs miRNA:

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3. Branched Chain Amino Acids (BCAAs)

Special Features:

• The only amino acids metabolized primarily in skeletal muscle (not liver)
• Liver has very low BCAA transaminase activity
• Major fuel for muscle during fasting, exercise, and trauma
• Provide nitrogen for glutamine and alanine synthesis

Common Catabolic Pathway (First Two Steps Shared):

Step 1: TRANSAMINATION
BCAAs + α-Ketoglutarate
        ↓ BCAA Transaminase (requires Pyridoxal phosphate, B6)
α-Keto acids (BCKAs) + Glutamate

Step 2: OXIDATIVE DECARBOXYLATION (rate-limiting)
BCKAs + CoA + NAD⁺
        ↓ BCKDH Complex (Branched-Chain Keto Acid Dehydrogenase)
           (requires: TPP, Lipoate, FAD, NAD⁺, CoA - same as PDH)
Acyl-CoA derivatives + CO₂ + NADH

Individual Fate (After Common Steps):

Role in Inter-organ Nitrogen Transport:

Muscle: Glucose → Pyruvate
        BCAAs donate amino group to pyruvate → ALANINE
        Alanine released into blood → travels to Liver
Liver: Alanine → Pyruvate + NH₃ (→ Urea)
       Pyruvate → Glucose (gluconeogenesis) → back to muscle

BCAAs → transamination → Glutamate → Glutamine (via glutamine synthetase)
Glutamine carried to liver, kidney, gut for nitrogen disposal

BCAA Deficiency Disease:

MSUD - Maple Syrup Urine Disease:

• Defect: BCKDH complex (branched-chain keto acid dehydrogenase) - deficient
• Inheritance: Autosomal recessive
• Accumulation: Leucine, Isoleucine, Valine + their keto acids
• Features: Sweet maple syrup odor in urine and cerumen; encephalopathy, seizures, mental retardation
• Treatment: BCAA-restricted diet (especially Leucine); thiamine supplementation; liver transplant
• Toxic metabolite: Leucine most neurotoxic

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4. Hyperuricaemia

Causes of Hyperuricaemia:

1. Underexcretion (>90% of cases):

2. Overproduction (<10% of cases):

Consequences (Clinical Features of Gout):

Hyperuricaemia
      ↓
Monosodium Urate (MSU) crystal deposition
      ├── JOINTS → ACUTE GOUTY ARTHRITIS
      │    (podagra = 1st MTP joint; needle-shaped, negatively birefringent crystals)
      │
      ├── SOFT TISSUES → TOPHI
      │    (ear pinna, tendons, olecranon bursa)
      │
      └── KIDNEY → URIC ACID NEPHROLITHIASIS
           (radiolucent stones on X-ray)

Key exam fact: Diagnosis confirmed by synovial fluid aspiration showing needle-shaped MSU crystals that are negatively birefringent under polarized light.

Treatment:

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5. Blotting Techniques

Complete Comparison Table:

Southern Blotting - Step by Step:

1. Extract DNA from cells (e.g., WBCs)
2. Cleave with Restriction Endonuclease → millions of fragments
3. Separate by Agarose Gel Electrophoresis (size-based)
4. Denature DNA (NaOH) → single stranded
5. Transfer (blot) to Nitrocellulose/Nylon Membrane
6. Hybridize with Radiolabeled (³²P) or fluorescent DNA Probe
7. Wash off unbound probe
8. Autoradiography → Bands revealed
   ↓
   Band pattern = specific DNA sequence identified

• Detection of gene mutations (large deletions, insertions)
• Trinucleotide repeat expansion (e.g., Fragile X, Huntington)
• RFLP analysis for genetic mapping
• Diagnosing infections (e.g., HIV proviral DNA)

Northern Blotting:

• Same as Southern but uses mRNA as target (in formaldehyde gel to denature RNA)
• Uses RNase inhibitors during RNA extraction
• Gives mRNA size AND quantity - unique advantage over RT-PCR
• Now largely replaced by RT-PCR and RNA sequencing

Western Blotting (Immunoblot):

• Proteins separated by SDS-PAGE (by molecular weight)
• Transferred to membrane → incubated with primary antibody → secondary antibody (enzyme/fluorescent labeled) → detection
• Confirmatory test for HIV (detects anti-HIV antibodies against specific viral proteins)
• Used for: protein identification, post-translational modifications, protein levels in disease

Memory Aid:

"Some Nights Will Shower Frequently"
Southern → DNA
Northern → RNA
Western → Protein
Southwestern → DNA-binding protein
Far-Western → Protein-protein interaction

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Sources: Guyton & Hall Medical Physiology; Lippincott Illustrated Reviews Biochemistry 8th Ed; Basic Medical Biochemistry - A Clinical Approach 6e; Harper's Illustrated Biochemistry 32nd Ed; Goldman-Cecil Medicine; Robbins & Cotran Pathologic Basis of Disease

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🧬 MBBS Biochemistry - Master Summary Sheet

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SESSION 1 TOPICS

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1. Regulation of Gene Expression

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2. Principle of Electrophoresis

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3. Northern Blotting

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SESSION 2 TOPICS

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4. Chromatography - Principle & Types

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5. Products Derived from Glycine

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6. Salient Features of Genetic Code

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7. Quality Control

• Levey-Jennings chart - daily QC plotted around mean ± SD
• Westgard rules - 1₂ₛ = warning; 1₃ₛ = reject; 2₂ₛ = systematic error; R₄ₛ = random error
• Internal QC = daily control sera (precision)
• External QC (EQA) = interlaboratory comparison (accuracy)
• CV% = (SD/Mean) × 100; good lab < 5%

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SESSION 3 TOPICS

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8. 25 Tumour Markers

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9. Gene Therapy

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10. Quaternary Structure of Protein

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11. Free Radicals

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SESSION 4 TOPICS

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12. Toxins - Causes & Deficiency

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13. miRNA & ncRNA

Gene → pri-miRNA → (Drosha/DGCR8) → pre-miRNA → Exportin-5 → 
Cytoplasm → DICER → mature miRNA → RISC → mRNA silenced

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14. Branched Chain Amino Acids

BCAA → (BCAA transaminase, B6) → BCKA → (BCKDH complex) → Acyl-CoA

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15. Hyperuricaemia

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16. Blotting Techniques

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QUICK REVISION TABLE - All 16 Topics

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Exam Tip: For 5-mark short answers in Vasudevan-based exams: Write definition → mechanism/pathway → table/diagram → clinical significance in every answer. This format reliably scores full marks.