Biotechnology — Principles and Processes

10 recombinant proteins used in medical practice: 1. Human Insulin (Humulin): • Produced in E. coli or Saccharomyces cerevisiae • Used to treat Type 1 (and Type 2) diabetes mellitus • Replaced animal insulin; first recombinant therapeutic protein (1982) 2. Human Growth Hormone (somatotropin, rHGH): • Produced in E. coli • Used to treat growth hormone deficiency in children (pituitary dwarfism) 3. Erythropoietin (EPO): • Produced in CHO (Chinese Hamster Ovary) cells • Stimulates red blood cell production • Used to treat anaemia in chronic kidney disease, cancer chemotherapy patients 4. Streptokinase (recombinant): • Clot-dissolving enzyme; produced by Streptococcus (original) • Modified recombinant version: used as thrombolytic (clot buster) in myocardial infarction and stroke 5. Interferon-α (IFN-α): • Produced in E. coli • Antiviral and anti-tumour activity • Used for hepatitis B, hepatitis C, some cancers (hairy cell leukaemia) 6. Tissue Plasminogen Activator (tPA): • Produced in CHO cells • Dissolves blood clots; used in acute myocardial infarction and ischaemic stroke 7. Factor VIII: • Clotting factor for haemophilia A treatment • Recombinant Factor VIII replaces blood-derived (risk of HIV, hepatitis transmission) 8. Factor IX: • Clotting factor for haemophilia B treatment 9. Hepatitis B Vaccine (HBsAg): • Surface antigen of HBV produced in yeast (Saccharomyces cerevisiae) • Used for vaccination against hepatitis B 10. Monoclonal antibodies (e.g., Herceptin/Trastuzumab): • Produced using hybridoma technology + recombinant methods • Trastuzumab: treats HER2-positive breast cancer • Others: Rituximab (lymphoma), Bevacizumab (anti-VEGF cancer), Adalimumab (arthritis)

Comparison of key molecular biology enzymes: Restriction Endonucleases (Restriction Enzymes): • Source: Bacteria (as a defence system against bacteriophages) • Function: Cuts (cleaves) DNA at specific recognition sequences (palindromic sequences, ~4–8 bp) • Action: Endonuclease — cuts within DNA; creates sticky ends or blunt ends • Example: EcoRI (from E. coli) cuts at 5'–GAATTC–3' • Used in: Recombinant DNA technology (restriction mapping, cloning) DNA Polymerase: • Source: All living organisms (DNA Pol I, II, III in prokaryotes; Pol α, β, δ, ε in eukaryotes) • Function: Synthesises new DNA strand using existing DNA as template • Action: Adds deoxyribonucleotides in 5'→3' direction; requires primer • Cannot initiate new strand; also has proofreading activity (3'→5' exonuclease) • Used in: DNA replication, PCR (Taq polymerase) RNA Polymerase: • Source: All living organisms • Function: Synthesises RNA from DNA template (transcription) • Action: Reads DNA template 3'→5'; synthesises RNA in 5'→3' direction • Does NOT require a primer; can initiate new strand • Ribonucleotides (ATP, GTP, CTP, UTP) are used • Used in: Transcription; in vitro RNA synthesis DNA Ligase: • Source: All living organisms (T4 DNA ligase commonly used in labs) • Function: Joins (seals) nicks in DNA; forms phosphodiester bonds between adjacent nucleotides • Action: Joins Okazaki fragments; seals recombinant DNA molecules after restriction enzyme cutting • Requires: ATP (or NAD⁺ in E. coli) as cofactor • Used in: DNA repair, replication, recombinant DNA technology Similarities among all four: • All are enzymes (biological catalysts — proteins) • All act on nucleic acids (DNA or RNA) • All require divalent metal ions (Mg²⁺) as cofactors (except ligase which uses ATP) • All are essential for molecular biology research and biotechnology

Two classic experiments proved DNA is the genetic material: Experiment 1: Griffith's Transformation Experiment (1928): • Two strains of Streptococcus pneumoniae: – Smooth (S) strain: Has polysaccharide capsule → virulent (causes pneumonia) – Rough (R) strain: No capsule → non-virulent (mice survive) Experiment: (a) Live S strain injected into mice → mice die (b) Live R strain injected → mice survive (c) Heat-killed S strain injected → mice survive (dead bacteria are harmless) (d) Heat-killed S + Live R injected → mice DIE; live S bacteria recovered from dead mice! Conclusion: Something from heat-killed S cells 'transformed' R cells into virulent S cells. This 'transforming principle' was a chemical substance. Experiment 2: Avery, MacLeod and McCarty (1944): • Biochemically identified Griffith's 'transforming principle' • Extracted different molecules from S cells: – DNA, RNA, proteins, lipids, polysaccharides • Treated each with specific enzymes: – DNase (destroys DNA): transformation ABOLISHED — no S colonies formed – RNase (destroys RNA): transformation still occurred – Protease (destroys proteins): transformation still occurred Conclusion: DNA — and DNA alone — is the transforming principle = genetic material Hershey-Chase Experiment (1952) — confirmed with bacteriophage: • T2 phage labelled with ³⁵S (protein) or ³²P (DNA) • Only ³²P (DNA) entered bacteria → only DNA carried genetic information into cells Conclusion: DNA is the genetic material.

(a) Plasmid DNA vs Chromosomal DNA: Plasmid DNA: • Small, circular, double-stranded DNA • Extrachromosomal — exists separately from the main chromosome • Typically 1–200 kb in size • Present mainly in bacteria (also in some yeast and plants) • Non-essential for normal growth under standard conditions (but may carry useful genes — antibiotic resistance, virulence factors) • Replicates independently using host cell machinery (has its own ori — origin of replication) • Can be transferred between bacteria by conjugation, transformation, or transduction • Used extensively as vectors in recombinant DNA technology Chromosomal DNA: • Large, linear (eukaryotes) or circular (prokaryotes) DNA • Main repository of genetic information for the organism • Contains all essential genes • Tightly packaged with proteins (histones in eukaryotes → chromatin → chromosomes) • Replication is coordinated with cell division • Prokaryotic chromosome: ~4.6 million bp (E. coli); Eukaryotic: hundreds of millions to billions bp (b) Genomic Library vs cDNA Library: Genomic Library: • Contains fragments of the ENTIRE genome of an organism (including all coding and non-coding sequences) • Made from: Total cellular DNA, digested with restriction enzymes → cloned into vectors • Includes: Exons, introns, promoters, regulatory regions, repetitive DNA, non-coding sequences • Represents complete genetic information • Used for: Chromosome mapping, studying gene regulation, cloning complete genes including introns • Much larger than cDNA library cDNA Library: • Contains only sequences derived from mRNA (expressed genes) in a particular cell/tissue/stage • Made from: mRNA extracted from cells → converted to cDNA using reverse transcriptase → cloned • Contains ONLY coding sequences (exons) — no introns, no regulatory regions • Tissue-specific: different tissues at different developmental stages have different cDNA libraries • Used for: Expressing eukaryotic genes in prokaryotes (E. coli lacks splicing machinery) • Smaller and simpler than genomic library • Used for: Studying expressed genes, producing recombinant proteins

PCR (Polymerase Chain Reaction): • A technique to amplify (make millions of copies of) a specific DNA sequence in vitro (in a test tube) • Developed by Kary Mullis (1983) — Nobel Prize in Chemistry 1993 Requirements: • Template DNA: The DNA to be amplified • Primers: Two short synthetic oligonucleotide sequences (~20 bp) complementary to either end of the target sequence • Taq DNA polymerase: Thermostable DNA polymerase from Thermus aquaticus (survives high temperatures) • dNTPs (deoxynucleoside triphosphates): Building blocks for new DNA • Buffer with Mg²⁺ ions • Thermal cycler (PCR machine) Process — 3 steps, repeated 30–40 times: Step 1 — Denaturation (94–96°C, ~1 min): • High temperature breaks hydrogen bonds between DNA strands • Double-stranded DNA → single strands Step 2 — Annealing (50–65°C, ~30 sec): • Temperature lowered • Primers bind (anneal) to complementary sequences on each single strand • Primer defines the region to be amplified Step 3 — Extension/Elongation (72°C, ~1 min/kb): • Taq polymerase extends primers in 5'→3' direction • New DNA strands synthesised using each single strand as template • 72°C is optimal for Taq polymerase Result: • Each cycle doubles the number of copies • 30 cycles → 2³⁰ ≈ 10⁹ copies of target DNA • Exponential amplification in a few hours Applications: • Medical diagnosis (HIV, TB, COVID-19 RT-PCR) • DNA fingerprinting (forensics, paternity testing) • Research: cloning, sequencing, gene expression studies • Archaeological DNA analysis • Prenatal diagnosis of genetic diseases

Gel Electrophoresis: • A technique to separate charged molecules (DNA, RNA, proteins) based on size and charge by passing electric current through a gel matrix Principle: • DNA is negatively charged (due to phosphate groups) → migrates toward positive electrode (anode) in an electric field • Smaller molecules move faster through the gel pores than larger molecules • Separation is based on size (molecular weight) Types: 1. Agarose gel electrophoresis: For separating DNA/RNA fragments 2. Polyacrylamide gel electrophoresis (PAGE): For proteins and smaller DNA/RNA 3. SDS-PAGE: For proteins denatured by SDS (sodium dodecyl sulphate) — separates by size only Procedure for DNA: 1. Agarose gel prepared (0.8–2% agarose in TAE or TBE buffer) 2. DNA samples mixed with loading dye (contains glycerol for density + dye for visualisation) 3. Samples loaded into wells in the gel 4. Electric current applied (100V for ~45 min) 5. DNA migrates toward positive pole; smaller fragments move farther 6. Gel stained with ethidium bromide (EtBr) or SYBR Green 7. DNA bands visualised under UV light 8. Size determined by comparison with DNA ladder (known size standards) Significance in biotechnology: • Verification of restriction enzyme digestion: confirms correct fragments produced • Checking PCR products: verify amplification • Southern blotting: gel → blot → probe hybridisation for gene detection • DNA fingerprinting: VNTR pattern separation • Quality control of plasmid preparations • Checking cloning results • Gene mapping and genome analysis • Diagnosis of genetic diseases (size of bands indicates mutations)