Semester 6
BP605T
Cloning Vectors, rDNA Technology & Recombinant Products
This unit is the heart of pharmaceutical biotechnology. It covers the molecular tools of genetic engineering — cloning vectors (plasmids, phages, cosmids), restriction endonucleases (molecular scissors), and DNA ligase. The rDNA technology workflow is studied step-by-step, followed by its landmark pharmaceutical applications: production of recombinant Interferon, Hepatitis-B vaccine, and Human Insulin. The unit concludes with PCR — the most transformative technique in molecular biology.
Syllabus & Topics
- 1Cloning Vectors – Requirements: A cloning vector is a DNA molecule used to carry foreign DNA into a host cell for replication. Essential features: (1) Origin of replication (ori): enables autonomous replication in host. (2) Selectable marker: gene conferring antibiotic resistance (Ampᴿ, Tetᴿ, Kanᴿ) → allows selection of transformed cells. (3) Multiple Cloning Site (MCS/polylinker): cluster of unique restriction enzyme sites for inserting foreign DNA. (4) Small size: easier manipulation and higher transformation efficiency. (5) High copy number (for amplification) or controlled expression (for protein production).
- 2Types of Cloning Vectors: (1) Plasmids: small (2-10 kb), circular, self-replicating DNA in bacteria. pBR322: first widely used — Ampᴿ + Tetᴿ markers, multiple RE sites, ~4.4 kb. pUC vectors: improved — lacZ gene for blue-white screening (insertional inactivation), higher copy number, smaller size. (2) Bacteriophage λ: phage DNA (~48 kb), can accommodate 15-20 kb inserts (larger than plasmids). λgt10, λgt11 for genomic/cDNA libraries. (3) Cosmids: hybrid of plasmid + phage λ cos sites. Package in phage particles but replicate as plasmids. Accept 35-45 kb inserts. (4) BACs (Bacterial Artificial Chromosomes): 100-300 kb inserts — for genome projects. (5) YACs (Yeast Artificial Chromosomes): up to 1 Mb — largest inserts.
- 3Restriction Endonucleases: ‘Molecular scissors’ — enzymes that recognize specific palindromic DNA sequences (4-8 bp) and cleave both strands. Type II restriction enzymes are most useful (cleave at or near recognition site). Examples: EcoRI (E. coli): 5′-G↓AATTC-3′ → sticky ends (5′ overhang). BamHI: 5′-G↓GATCC-3′ → sticky ends. HindIII: 5′-A↓AGCTT-3′ → sticky ends. SmaI: 5′-CCC↓GGG-3′ → blunt ends. Sticky (cohesive) ends: single-stranded overhangs → facilitate ligation (base-pair with complementary sticky ends). Blunt ends: no overhangs → can ligate with any blunt end (less efficient). Nomenclature: Eco (E. coli), R (strain RY13), I (first enzyme discovered).
- 4DNA Ligase: ‘Molecular glue’ — joins DNA fragments by forming phosphodiester bonds between 3′-OH and 5′-phosphate groups. T4 DNA ligase (from bacteriophage T4): most commonly used — can ligate both sticky AND blunt ends (blunt end ligation requires higher enzyme concentration). E. coli DNA ligase: ligates only sticky/nick ends, requires NAD⁺. Ligation reaction: foreign DNA (insert with compatible sticky ends) + linearized vector (cut with same RE) + T4 DNA ligase → recombinant DNA molecule.
- 5Recombinant DNA Technology – Steps: Step 1: Gene identification and isolation (from genomic DNA, cDNA library, or chemical synthesis). Step 2: Insert gene into vector (cut both with same RE → ligate with DNA ligase). Step 3: Transform vector into host (E. coli, yeast, or mammalian cells). Transformation methods: heat shock (CaCl₂), electroporation, chemical (lipofection for mammalian). Step 4: Selection of recombinants (antibiotic resistance + blue-white screening or reporter genes). Step 5: Screening and confirmation (colony PCR, restriction analysis, sequencing). Step 6: Expression of recombinant protein (inducible promoter system — IPTG/lac, T7 promoter). Step 7: Purification of protein (chromatography — affinity, ion-exchange, size-exclusion).
- 6Applications of Genetic Engineering in Medicine: (1) Recombinant protein drugs: Insulin, Interferon, Growth Hormone, EPO, Factor VIII, tPA. (2) Recombinant vaccines: Hepatitis B (HBsAg in yeast), HPV vaccine. (3) Gene therapy: correcting genetic defects (ADA-SCID, β-thalassemia). (4) Diagnostic tools: PCR (COVID-19 testing), DNA probes, recombinant antigens for ELISA. (5) Pharmacogenomics: tailoring drug therapy based on genetic profile. (6) Transgenic animals: drug testing, production of therapeutic proteins in milk (pharming). (7) Monoclonal antibodies: engineered antibodies for cancer, autoimmune diseases.
- 7Recombinant Interferon: Interferons (IFNs): cytokines with antiviral, antiproliferative, and immunomodulatory activity. Types: IFN-α (leukocyte — antiviral, anti-cancer), IFN-β (fibroblast — MS), IFN-γ (T-cell — immunostimulant). Production by rDNA: (1) IFN-α gene isolated from human leukocyte mRNA → cDNA by reverse transcriptase. (2) cDNA inserted into E. coli expression vector. (3) IFN-α expressed as inclusion bodies → solubilized, refolded, purified. (4) PEGylation (attachment of PEG) → PEG-Interferon-α (Pegasys) → longer t½, once-weekly dosing. Uses: Hepatitis B & C (now largely replaced by DAAs for HCV), hairy cell leukemia, melanoma, CML.
- 8Recombinant Hepatitis B Vaccine: First recombinant vaccine approved (1986). Production: (1) HBsAg gene (surface antigen gene of Hepatitis B virus) cloned into yeast expression vector (Saccharomyces cerevisiae). (2) Yeast expresses HBsAg → self-assembles into 22 nm virus-like particles (VLPs) that are immunogenic but NON-INFECTIOUS (no viral DNA → cannot cause infection). (3) VLPs purified → adsorbed onto aluminum hydroxide adjuvant. Advantages over plasma-derived vaccine: unlimited supply, no risk of transmitting blood-borne infections, consistent quality. Given as 3-dose schedule (0, 1, 6 months). Part of Universal Immunization Programme (India).
- 9Recombinant Human Insulin (Humulin): Before rDNA: insulin extracted from pig/cow pancreas → immunogenicity (anti-insulin antibodies). Recombinant production — Two approaches: (1) Two-chain method (Genentech, 1978): A-chain (21 aa) and B-chain (30 aa) genes synthesized separately → expressed in E. coli as fusion proteins with β-galactosidase → chains purified → joined by oxidative sulfhydryl coupling (disulfide bonds). First recombinant pharmaceutical product approved (1982). (2) Proinsulin method (Eli Lilly): single proinsulin gene → expressed in E. coli → proinsulin refolded → C-peptide cleaved by trypsin + carboxypeptidase → mature insulin. More efficient (correct disulfide bonds formed naturally during proinsulin folding). Insulin analogs: Lispro (rapid-acting), Glargine (long-acting) — engineered by amino acid substitutions.
- 10Polymerase Chain Reaction (PCR): Invented by Kary Mullis (Nobel Prize, 1993). PCR amplifies a specific DNA sequence exponentially — from a few copies to millions in hours. Components: (1) Template DNA (the DNA containing the target sequence). (2) Two primers (short oligonucleotides, ~18-25 bp, flanking the target sequence). (3) Taq DNA polymerase (thermostable polymerase from Thermus aquaticus — survives 95°C). (4) dNTPs (deoxynucleotide triphosphates — building blocks). (5) Buffer with Mg²⁺.
- 11PCR – Steps & Applications: Three steps per cycle (repeated 25-35 cycles): (1) Denaturation (94-95°C, 30 sec): DNA strands separate. (2) Annealing (50-65°C, 30 sec): primers bind to complementary sequences on template. (3) Extension (72°C, 1-2 min): Taq polymerase extends primers → synthesizes new strands. After n cycles: 2ⁿ copies (30 cycles → ~10⁹ copies). Applications: (1) Diagnostics: pathogen detection (COVID-19 RT-PCR, TB, HIV viral load). (2) Forensics: DNA fingerprinting from minute samples. (3) Genetic testing: mutation detection, prenatal diagnosis. (4) Cloning: gene amplification before insertion into vector. (5) Archaeology: amplifying ancient DNA. Variants: RT-PCR (reverse transcriptase — for RNA), Quantitative/Real-time PCR (qPCR — quantifies DNA in real-time), Multiplex PCR (multiple targets simultaneously).
Learning Objectives
Vector Features: List the essential features of a good cloning vector and compare plasmids, phages, and cosmids.
RE Nomenclature: Explain restriction enzyme nomenclature and differentiate sticky vs blunt end cuts with examples.
rDNA Steps: Describe the complete step-by-step process of recombinant DNA technology.
Insulin Production: Draw the flowchart for recombinant human insulin production by the proinsulin method.
PCR Steps: Describe the three steps of PCR and explain the role of Taq polymerase.
Exam Prep Questions
Q1. Why can’t we use normal DNA polymerase in PCR?
PCR requires heating to 94–95°C for denaturation in EVERY cycle (25–35 cycles). Normal DNA polymerases (from E. coli or humans) are denatured (destroyed) at this temperature. Taq polymerase, isolated from Thermus aquaticus (a bacterium living in hot springs at 70–80°C), is thermostable — it retains activity even after repeated exposure to 95°C. Without Taq, you would need to add fresh polymerase after every denaturation step — making PCR impractical. This discovery is what made PCR a practical laboratory technique.
Q2. Why is the Hepatitis B vaccine produced in yeast rather than E. coli?
HBsAg requires post-translational modifications (glycosylation, proper folding) and must self-assemble into virus-like particles (VLPs) to be immunogenic. E. coli is a prokaryote → cannot glycosylate proteins or form VLPs properly. Saccharomyces cerevisiae (yeast) is a eukaryote → can perform glycosylation, proper protein folding, and allows HBsAg to assemble into 22 nm VLPs that mimic the actual viral surface → strong immune response without any risk of infection.
Q3. What is Blue-White Screening?
Blue-white screening identifies recombinant clones (containing insert) from non-recombinant (empty vector). The vector has the lacZ gene (encoding β-galactosidase) with the MCS inside it. When foreign DNA is inserted into the MCS → lacZ is disrupted → no functional β-galactosidase → WHITE colonies (on X-gal + IPTG plates). Empty vector: intact lacZ → β-galactosidase produced → cleaves X-gal → BLUE colonies. So: White = recombinant (pick these), Blue = non-recombinant (discard).