Home › Biology › Q
BiologyMolecular Biology / Biotechnology
Which of the following statements are CORRECT regarding recombinant DNA technology?
A. Restriction endonucleases cut DNA at specific palindromic sequences.
B. DNA ligase joins two DNA molecules by forming peptide bonds.
C. Plasmids are used as vectors in gene cloning.
D. PCR amplifies RNA directly without any intermediate step.
Options
1
A and B only
2
B and D only
3
A and C only
4
C and D only
Correct Answer
A and C only
Solution
1

A: Restriction endonucleases cut at palindromic sequences = TRUE

B: DNA ligase forms phosphodiester bonds (NOT peptide bonds) = FALSE

2

C: Plasmids used as cloning vectors = TRUE

D: PCR amplifies DNA, not RNA directly (need RT-PCR for RNA) = FALSE

Answer: A and C only

A: TRUE (palindromic cuts) | B: FALSE (phosphodiester not peptide) | C: TRUE (plasmid vector) | D: FALSE (PCR = DNA, not RNA)
Theory: Molecular Biology / Biotechnology
1. Recombinant DNA Technology Overview

Recombinant DNA technology: combining DNA from different sources. Key tools: Restriction endonucleases (molecular scissors), DNA ligase (molecular glue), vectors (carriers), host cells, PCR (amplification). Applications: insulin production (1982, first recombinant protein), growth hormone, vaccines (hepatitis B), transgenic plants (Bt cotton), gene therapy, diagnostics. History: 1970 — Hamilton Smith isolated first restriction enzyme. 1972 — Paul Berg made first recombinant DNA molecule (Nobel 1980). 1973 — Cohen and Boyer first cloned foreign gene in bacteria. 1982 — Eli Lilly launched Humulin (recombinant human insulin). 1985 — Kary Mullis invented PCR (Nobel 1993).

2. Restriction Endonucleases

Type I: cut DNA ~1000 bp from recognition site. Random cutting. Not used in cloning. Type II: cut at or near specific recognition sequences. Used in recombinant DNA technology. Type III: intermediate. Type II examples: EcoRI (E. coli, recognition sequence GAATTC, produces 5-AATT sticky ends). BamHI (recognition GGATCC, 5-GATC sticky ends). HindIII (recognition AAGCTT, 5-AGCT sticky ends). SmaI (recognition CCCGGG, blunt ends). EcoRV (recognition GATATC, blunt ends). Palindromic sequences: same 5-to-3 sequence on both strands. GAATTC on one strand reads same as complement read 5-to-3. Sticky ends: single-stranded overhangs (5 or 3). Enable complementary joining of fragments from different sources. Blunt ends: no overhang. Less efficient ligation. Isoschizomers: different enzymes recognising same sequence.

3. Vectors for Gene Cloning

Vector: DNA molecule used to carry foreign DNA into host cell. Requirements: origin of replication (ori), selectable marker (antibiotic resistance gene), unique restriction sites (MCS = multiple cloning site). Plasmid vectors: pBR322 (first artificial plasmid, ampicillin + tetracycline resistance). pUC19 (blue-white screening). pGEX (GST fusion protein production). pET (T7 promoter, high expression). Bacteriophage vectors: lambda phage, M13. Can carry larger inserts than plasmids. Cosmids: plasmid + phage cos sites. Carry 40-50 kb inserts. BAC (bacterial artificial chromosome): carry 100-350 kb. Used in genome projects. YAC (yeast artificial chromosome): carry 100-2000 kb. Used for large genomic fragments. Shuttle vectors: replicate in two different host organisms (E. coli + yeast). Ti plasmid (Agrobacterium tumefaciens): natural vector for plant transformation.

4. PCR - Polymerase Chain Reaction

PCR: amplifies specific DNA sequences exponentially in vitro. Invented by Kary Mullis, 1985 (Nobel 1993). Three steps per cycle: Denaturation: 94-98°C, separates double-stranded DNA. Annealing: 50-65°C, primers bind to complementary sequences on template. Extension: 72°C, Taq polymerase extends primers (adds dNTPs). Taq polymerase: from thermophilic bacterium Thermus aquaticus. Heat-stable. Pfu polymerase: higher fidelity. After 30 cycles: 2^30 = ~10^9 copies from 1 template molecule. Components: Template DNA, two primers (20-25 nt), dNTPs (dATP, dCTP, dGTP, dTTP), DNA polymerase (Taq), Mg2+ (cofactor), buffer. RT-PCR: for RNA. Reverse transcriptase first converts RNA to cDNA, then PCR amplifies cDNA. Used for: mRNA expression analysis, detecting RNA viruses (COVID-19 diagnostic). Quantitative PCR (qPCR): measures amount of PCR product in real time using fluorescent dye or probe.

5. Cloning Strategies

Directional cloning: use two different restriction enzymes on vector and insert. Insert can only go in one orientation. Blue-white screening: vector contains lacZ gene (beta-galactosidase) with MCS inserted in it. Successful insert disrupts lacZ. Blue colonies: no insert (lacZ functional, cleaves X-gal → blue). White colonies: insert present (lacZ disrupted, no blue colour). Antibiotic selection: vector carries ampicillin resistance. Transformed bacteria: grow on ampicillin plates (untransformed die). Insertional inactivation: if insert disrupts second marker (e.g., tetracycline resistance), colonies sensitive to tetracycline but resistant to ampicillin = have vector with insert. Replica plating: identify colonies with insert vs empty vector. Expression cloning: insert under control of promoter → express foreign protein in host. His-tag, GST-tag: purification affinity tags added to recombinant protein.

6. DNA Blotting Techniques

Southern blot (Ed Southern, 1975): DNA fragments separated by gel electrophoresis → transferred to nitrocellulose/nylon membrane → hybridised with labelled DNA probe → detect specific DNA sequence. Used for: RFLP analysis, gene detection. Northern blot: RNA (not DNA) → probe detects specific mRNA → gene expression analysis. Western blot: proteins separated by SDS-PAGE → transferred to membrane → detected by specific antibody (primary) + secondary antibody with enzyme/fluorescent label. Diagnose: HIV (Western blot confirms ELISA positive), Lyme disease, BSE. Eastern blot: detecting post-translational modifications. Southwestern: detect DNA-binding proteins. Far-Western: detect protein-protein interactions. FISH (Fluorescence in situ hybridisation): fluorescent probes hybridise to specific chromosomal regions. Detect chromosomal aberrations, gene localisation.

7. Bioinformatics and Genomics

Genome: complete set of genetic material. Human genome: ~3.2 billion bp, ~20,000-25,000 protein-coding genes, ~1.5% protein-coding, rest = introns, regulatory sequences, transposons, pseudogenes. Human Genome Project (HGP): 1990-2003. International collaboration. Shotgun sequencing approach. Impact: disease gene identification, pharmacogenomics, personalised medicine. Bioinformatics: computational analysis of biological data. Databases: GenBank (DNA sequences), UniProt (proteins), PDB (protein structures). BLAST: compare sequences to database (find similar sequences, predict function). Phylogenetics: evolutionary relationships from sequence data. Comparative genomics: compare genomes between species. CRISPR-Cas9: revolutionary genome editing. Guide RNA + Cas9 → precise DNA cuts → gene editing. Nobel Prize 2020 (Doudna, Charpentier). Applications: sickle cell disease treatment (approved 2023), cancer immunotherapy, agriculture.

8. Applications of Biotechnology

Insulin production: human insulin gene inserted into E. coli or Saccharomyces cerevisiae via plasmid. Recombinant human insulin (Humulin) approved 1982. Before: pig/cow insulin used (slightly different structure, could cause immune reactions). Growth hormone: recombinant human GH (somatropin) for children with GH deficiency. Before: extracted from cadaver pituitary (prion disease risk). Vaccines: Hepatitis B vaccine: HBsAg (surface antigen) produced in yeast. No live virus. Very safe. HPV vaccine: virus-like particles (VLPs) from yeast or insect cells. Bt crops: Bacillus thuringiensis cry genes inserted into crop plants. Cry proteins toxic to specific insects (lepidopteran larvae, beetles) but safe for humans. Golden rice: rice engineered to produce beta-carotene (vitamin A precursor) in endosperm. Biopharmaceuticals: ~250 recombinant proteins on market. Monoclonal antibodies (mAbs): rituximab, trastuzumab, adalimumab, infliximab, bevacizumab. Gene therapy: replace/correct defective genes. ADA-SCID (first gene therapy success, 1990). SMA (spinal muscular atrophy): Zolgensma (AAV-delivered SMN1 gene) = most expensive drug ever.

Frequently Asked Questions
1. What are the key properties that make restriction enzymes useful for recombinant DNA? ⌄
Type II restriction enzymes are invaluable because: (1) Sequence specificity: each enzyme recognises a specific palindromic sequence (usually 4, 6, or 8 bp). 6-cutter enzymes (like EcoRI, BamHI) cut roughly every 4000 bp in random sequence DNA (4^6 = 4096). This gives manageable-sized fragments. (2) Palindromic recognition: produces identical overhangs on both strands. (3) Sticky ends: the 5 or 3 overhangs left after cutting are complementary. Any two fragments cut with the same enzyme will have complementary sticky ends that can base-pair and be joined by DNA ligase. (4) Predictability: gives defined, reproducible fragments (RFLP - restriction fragment length polymorphism). (5) Availability: hundreds of enzymes now commercially available with different specificities. The ability to cut DNA at specific, reproducible sites and to rejoin fragments from different sources is the entire basis of recombinant DNA technology.
2. How does PCR work step by step and what are its applications? ⌄
PCR cycle (3 steps, repeated 30-40 times): Step 1 - Denaturation (94-98°C, 30 sec): heat breaks H-bonds between base pairs → two single-stranded templates. Step 2 - Annealing (50-65°C, 30 sec): temperature lowered → short oligonucleotide primers (20-25 nt) bind to complementary sequences flanking the target region. Primer design: Tm 55-65°C, no self-complementarity, unique to target. Step 3 - Extension (72°C, 1 min per kb): Taq polymerase extends primers in 5 to 3 direction, adding dNTPs complementary to template. Result: double-stranded copy of target region. After n cycles: 2^n copies. 30 cycles = 2^30 = ~10^9. Applications: diagnostics (COVID-19 test = RT-qPCR for SARS-CoV-2 RNA), forensics (DNA fingerprinting from tiny samples), ancestry testing, paternity, gene cloning, site-directed mutagenesis, gene expression (RT-qPCR), ancient DNA analysis.
3. Why cannot PCR directly amplify RNA? ⌄
DNA polymerase (including Taq polymerase used in PCR) requires a DNA template. RNA is a different chemical (ribose vs deoxyribose, single-stranded, uracil instead of thymine). Taq polymerase cannot use RNA as template. Solution for RNA: RT-PCR (Reverse Transcriptase PCR). Step 1: Reverse transcriptase (an RNA-dependent DNA polymerase from retroviruses like MMLV = Moloney murine leukaemia virus) copies RNA into complementary DNA (cDNA): mRNA + primer + reverse transcriptase + dNTPs → cDNA. Step 2: Regular PCR amplifies the cDNA. Why use RT-PCR? mRNA tells you which genes are actively expressed (transcription). cDNA lacks introns (easier to express in bacteria). Diagnose RNA viruses (HIV, influenza, SARS-CoV-2). Quantitative RT-PCR (RT-qPCR): measures the amount of specific mRNA = gene expression analysis. Essential tool in molecular biology research.
4. What is the difference between gene cloning and PCR for amplifying a gene? ⌄
Gene cloning: insert gene into vector → transform into host bacteria → bacteria multiply and carry the gene → extract plasmid + insert. Time: days to weeks. Requires: restriction enzymes, ligase, vector, competent bacteria. Produces: unlimited quantities of gene in stable form (can store bacteria). Can express the gene (make protein) if proper promoter present. PCR: amplify gene directly in vitro. Time: 3-4 hours. Requires: purified template DNA, primers, Taq polymerase, dNTPs, thermocycler. Produces: DNA fragment (no vector, not in living cells). Cannot directly express protein from PCR product without cloning into expression vector. Advantages of each: PCR = fast, simple, small template. Cloning = stable, expressible, unlimited bacteria. Modern approach: PCR first to amplify the gene → then clone PCR product into vector → verify by sequencing → express protein. PCR + cloning are complementary, not alternative, techniques.
5. How are transgenic plants and animals created? ⌄
Transgenic plants: (1) Agrobacterium tumefaciens method: Agrobacterium is a soil bacterium with Ti (tumour-inducing) plasmid. T-DNA region of Ti plasmid naturally integrates into plant nuclear genome when bacterium infects wounded plant. Engineered: remove tumour-causing genes from T-DNA, insert gene of interest. Agrobacterium delivers gene of interest into plant cells. Plant cells regenerated into transgenic plant. Works well for dicots (tomato, tobacco, potato, cotton). (2) Biolistics (gene gun): gold or tungsten particles coated with DNA shot into plant cells using helium gas pressure. Works for monocots (wheat, maize, rice). (3) Electroporation of protoplasts. Transgenic animals: (1) Microinjection: inject DNA into pronucleus of fertilised egg → embryo implanted into foster mother. Random integration into genome. (2) ES cell method: introduce gene by homologous recombination into embryonic stem cells → inject into blastocyst → chimeric animal → breeding gives transgenic offspring. Used for gene knockouts (gene targeting). (3) CRISPR: modify zygote directly. Precise gene editing.
Previous Questions
Q.
ADH antidiuretic hormone vasopressin posterior pituitary collecting duct AQP2 water
Biology . ADH Vasopressin
Q.
Rho factor transcription termination prokaryotes RNA helicase rut site
Biology . Termination of transcription
Q.
Innate immunity NK cells complement proteins C D only not antibody T cell
Biology . C and D only
Q.
Apoenzyme protein component holoenzyme cofactor coenzyme prosthetic group
Biology . Apoenzyme
Q.
Digestive enzymes pepsin lipase amylase dipeptidase A-IV B-II C-I D-III
Biology . A-IV B-II C-I D-III