Biotechnology — Principles and Processes
Welcome to HSLC Guru! This page provides comprehensive question answers for Class 12 Biology Chapter 11 — Biotechnology: Principles and Processes for ASSEB (Assam State School Education Board) students. Biotechnology has revolutionised medicine, agriculture, and industry through genetic engineering and bioprocess engineering. This chapter explains the principles, tools, and processes of recombinant DNA technology — including restriction enzymes, vectors, PCR, and bioreactors. Our notes are crafted strictly for the ASSEB Class 12 Biology syllabus, with detailed summary, Q&A in 1-mark, 2–3-mark, and 5–7-mark formats, MCQs, fill-in-the-blanks, true/false questions, and a glossary to make exam preparation thorough and easy.
Summary
Biotechnology is the use of living organisms, cells, or biological processes to manufacture products useful to humans. According to the European Federation of Biotechnology (EFB), biotechnology is defined as “the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.” Traditional biotechnology includes age-old practices such as fermentation in making curd, bread, wine, beer, and cheese using microorganisms. Modern biotechnology, on the other hand, makes use of genetically modified organisms, recombinant DNA technology, tissue culture, and bioprocess engineering to produce vaccines, hormones (insulin), antibiotics, transgenic crops, and biofuels at industrial scale.
Modern biotechnology rests on two core principles. The first is genetic engineering — techniques to alter the genetic material (DNA/RNA) of an organism, introduce it into another host, and change the host’s phenotype. The second is bioprocess engineering — the maintenance of a sterile (microbial-contamination-free) ambience in chemical engineering processes to enable the growth of only the desired microbe/eukaryotic cells in large quantities for the production of biotechnological products like antibiotics, vaccines, and enzymes. Together these principles allow controlled manipulation of genes and large-scale production of useful biomolecules.
The tools of recombinant DNA technology include restriction enzymes, DNA ligase, vectors, competent host cells, gel electrophoresis equipment, and PCR machines. Restriction enzymes are molecular scissors that cut DNA at specific recognition sequences. The first restriction endonuclease, Hind II, was isolated by Smith and Nathans; EcoRI, a widely used enzyme, was isolated from Escherichia coli RY13. They recognise palindromic sequences (sequences that read the same on both strands when read in the 5’→3′ direction) and produce sticky ends with short single-stranded overhangs that allow easy joining. DNA ligase seals the nicks between sticky ends to form recombinant DNA. Vectors are carrier DNA molecules used to transfer foreign DNA into a host. Common vectors include plasmids (e.g., pBR322), bacteriophages (e.g., λ phage), cosmids, BACs and YACs (for large inserts), Ti plasmid of Agrobacterium tumefaciens for plants, and retroviral vectors for animal cells. Vectors carry selectable markers such as antibiotic resistance genes (ampr, tetr) which help differentiate transformants from non-transformants. The competent host commonly used is E. coli, made permeable to DNA by treatment with divalent cations (CaCl2) or by electroporation/heat shock.
Gel electrophoresis separates DNA fragments by size on an agarose gel under an electric field; DNA being negatively charged moves towards the anode. Bands are visualised under UV light after staining with ethidium bromide. Polymerase Chain Reaction (PCR), invented by Kary Mullis in 1983, amplifies a specific DNA segment exponentially using thermostable Taq DNA polymerase (from Thermus aquaticus) through repeated cycles of denaturation (94°C), annealing of primers (~54°C), and extension (72°C). The major processes in recombinant DNA technology are: (i) isolation of DNA from the source, (ii) cutting/restriction with specific enzymes, (iii) gel electrophoresis to separate and identify fragments, (iv) ligation of insert and vector to form recombinant DNA, (v) transformation/introduction into the competent host, (vi) selection of recombinants using markers, and (vii) culture in bioreactors (stirred-tank or sparged stirred-tank) followed by downstream processing — separation, purification, formulation, and quality control — to obtain a finished marketable biotech product.
1-Mark Questions
Q1. Define biotechnology as per EFB.
Answer: The European Federation of Biotechnology defines biotechnology as “the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”
Q2. Who isolated the first restriction enzyme?
Answer: Hamilton Smith and Daniel Nathans isolated the first restriction endonuclease (Hind II) in 1968.
Q3. Name the source organism of EcoRI.
Answer: EcoRI is isolated from the bacterium Escherichia coli strain RY13.
Q4. What is a palindromic sequence?
Answer: A palindromic sequence is a stretch of DNA that reads the same on both strands when read in the same (5’→3′) direction.
Q5. Who invented PCR and in which year?
Answer: Polymerase Chain Reaction (PCR) was invented by Kary Mullis in 1983.
Q6. Name the source of Taq DNA polymerase.
Answer: Taq DNA polymerase is obtained from the thermophilic bacterium Thermus aquaticus.
Q7. What is the role of DNA ligase?
Answer: DNA ligase seals the nicks between adjacent nucleotides by forming phosphodiester bonds, joining the insert DNA to the vector to form recombinant DNA.
Q8. Name two selectable markers used in pBR322.
Answer: The selectable markers in pBR322 are ampicillin resistance gene (ampr) and tetracycline resistance gene (tetr).
Q9. Which vector is used for gene transfer in plants?
Answer: The Ti plasmid of Agrobacterium tumefaciens is used as a vector for gene transfer in dicotyledonous plants.
Q10. What is downstream processing?
Answer: Downstream processing is the series of post-bioreactor steps — separation, purification, formulation with suitable preservatives, and quality control testing — used to make a biotech product market-ready.
2–3 Mark Questions
Q1. Differentiate between traditional biotechnology and modern biotechnology.
Answer: Traditional biotechnology refers to age-old fermentation practices using natural microbes — for example, curd, bread, wine, beer, and cheese making. It does not involve manipulation of the genetic material. Modern biotechnology makes use of genetically modified organisms, recombinant DNA technology, tissue culture, and bioprocess engineering to produce vaccines, hormones, transgenic crops, and biofuels at industrial scale, often involving deliberate gene transfer between species.
Q2. What are the two core principles of modern biotechnology?
Answer: The two core principles are: (i) Genetic engineering — techniques to alter the chemistry of genetic material (DNA/RNA), introduce it into a host, and change the host’s phenotype; and (ii) Bioprocess engineering — maintenance of sterile (contamination-free) conditions to allow only the desired microbe/eukaryotic cells to grow in large bioreactors for industrial production.
Q3. Explain the cutting action of EcoRI on a palindromic sequence.
Answer: EcoRI recognises the palindromic sequence 5′-GAATTC-3′ / 3′-CTTAAG-5′. It cleaves between G and A on each strand, producing fragments with single-stranded 5′ overhangs of “AATT”. These complementary overhangs are called sticky ends because they can re-anneal with any DNA fragment cut by the same enzyme, facilitating ligation by DNA ligase.
Q4. What features make a good cloning vector?
Answer: A good cloning vector should possess: (i) an origin of replication (ori) for autonomous replication; (ii) a selectable marker (e.g., antibiotic resistance gene) to identify transformants; (iii) unique restriction sites within the marker for inserting foreign DNA; and (iv) be of small size for easy entry into host cells. Higher copy number is desirable for greater yield.
Q5. Briefly describe gel electrophoresis.
Answer: Gel electrophoresis is a technique to separate DNA fragments based on size. The DNA samples are loaded into wells of an agarose gel and an electric current is applied. Negatively charged DNA migrates towards the anode; smaller fragments move faster than larger ones. After staining with ethidium bromide and exposure to UV light, the fragments appear as bright orange bands. The desired band is then cut out and eluted — a process called elution.
Q6. What is a competent host? How is competence achieved in E. coli?
Answer: A competent host is a cell that has been made temporarily permeable to foreign DNA so that recombinant DNA can be introduced. E. coli is the most commonly used host. Competence is induced by treating cells with a specific concentration of a divalent cation such as calcium chloride (CaCl2), followed by a brief heat shock at 42°C, or by using methods like electroporation or the gene gun (biolistics).
5–7 Mark Questions
Q1. Describe in detail the tools of recombinant DNA technology.
Answer: The tools of recombinant DNA technology include the following:
(i) Restriction enzymes: Molecular scissors which cleave DNA at specific palindromic recognition sequences. The first such enzyme, Hind II, was isolated by Smith and Nathans. EcoRI, isolated from E. coli RY13, recognises GAATTC and produces sticky ends.
(ii) DNA ligase: Joins the insert with the vector by forming phosphodiester bonds, sealing nicks and creating recombinant DNA.
(iii) Vectors: Carrier molecules to transfer foreign DNA into the host. Examples include plasmids (pBR322), bacteriophages (lambda), cosmids, BAC and YAC for large inserts, Ti plasmid for plants, and retroviral vectors for animal cells. They carry origin of replication, selectable markers (ampr, tetr) and recognition sites.
(iv) Competent host: Usually E. coli, treated with CaCl2 to make it permeable to DNA.
(v) Gel electrophoresis apparatus: For separating DNA fragments based on size. (vi) PCR machine (thermal cycler): For exponential amplification of the gene of interest. (vii) Bioreactors: For large-scale culture and production of recombinant products.
Q2. Describe the steps of recombinant DNA technology with a diagrammatic outline.
Answer: Recombinant DNA technology involves the following steps:
(i) Isolation of DNA: Cells are lysed using lysozyme (bacteria), cellulase (plants), or chitinase (fungi). RNA and proteins are removed by RNase and protease respectively. Pure DNA is precipitated using chilled ethanol.
(ii) Restriction (cutting): Both the source DNA and the vector are cut by the same restriction enzyme to give compatible sticky ends.
(iii) Gel electrophoresis: Used to separate fragments and confirm the cuts. The desired fragment is eluted from the gel.
(iv) Ligation: The gene of interest is joined to the vector by DNA ligase to form the recombinant DNA molecule.
(v) Transformation: The recombinant DNA is introduced into the competent host (E. coli) using CaCl2, heat shock, electroporation, microinjection, or a gene gun.
(vi) Selection of recombinants: Antibiotic markers and insertional inactivation are used to identify cells carrying the recombinant DNA.
(vii) Culture and downstream processing: Recombinant cells are cultured in bioreactors; the product is separated, purified, formulated, and quality tested before release to market.
Q3. Explain Polymerase Chain Reaction (PCR) and its three steps.
Answer: PCR was developed by Kary Mullis in 1983. It is an in-vitro technique to amplify a specific DNA segment exponentially using a pair of primers and the thermostable Taq DNA polymerase from Thermus aquaticus. Each cycle has three steps:
(i) Denaturation (94°C): The double-stranded template DNA is heated and the two strands separate.
(ii) Annealing (~54°C): The temperature is lowered, and two short oligonucleotide primers (forward and reverse) bind to their complementary regions on the single-stranded templates.
(iii) Extension (72°C): Taq DNA polymerase extends the primers using deoxynucleotide triphosphates (dNTPs), synthesising new complementary strands.
The cycle is repeated 25–35 times. As each new strand serves as a template in the next round, the target DNA is amplified exponentially — yielding about a billion copies in 30 cycles. PCR is widely used in DNA fingerprinting, diagnosis of genetic disorders, detection of pathogens (HIV, SARS-CoV-2), and forensic investigations.
Q4. Describe a bioreactor and the various downstream processing steps.
Answer: A bioreactor is a large vessel (100–1000 L or more) in which raw materials are biologically converted into specific products by microbes, plant or animal cells, or enzymes. Optimal growth conditions — temperature, pH, substrate, salt, vitamins, oxygen — are maintained.
Common types are: (i) Stirred-tank bioreactor — usually cylindrical, with a stirrer that mixes the contents and ensures even oxygen distribution; and (ii) Sparged stirred-tank bioreactor — air is bubbled through the medium (sparging) for better aeration. A bioreactor has an agitator system, oxygen delivery system, foam control, temperature and pH control, and sampling ports.
After biosynthesis, downstream processing includes: (i) Separation of cells/biomass from the medium; (ii) Purification using chromatography or filtration; (iii) Formulation with suitable preservatives or stabilisers; and (iv) Quality control (QC) testing for purity, potency, and safety. Only after passing QC is the product packaged and marketed.
Q5. Discuss the structure and significance of pBR322 as a cloning vector.
Answer: pBR322 is a popular E. coli cloning vector, named after its developers Bolivar and Rodriguez (“p” for plasmid, BR for Bolivar–Rodriguez, 322 is the lab number). It is a circular, double-stranded DNA molecule of about 4.36 kb. Important features include:
(i) ori — origin of replication, ensures autonomous replication and high copy number.
(ii) Two selectable marker genes: ampr (ampicillin resistance) and tetr (tetracycline resistance).
(iii) Several unique restriction sites (HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI) for inserting foreign DNA.
(iv) rop gene, which codes for a protein that helps in plasmid replication.
If foreign DNA is inserted at BamHI within tetr, the gene is inactivated (insertional inactivation). Recombinant cells then become tetracycline-sensitive but ampicillin-resistant, allowing easy identification on replica plates. Because of these well-defined features, pBR322 has been a standard teaching and research vector since 1977.
Multiple Choice Questions (MCQs)
Q1. The first restriction enzyme isolated was —
(a) EcoRI (b) Hind II (c) BamHI (d) HindIII
Answer: (b) Hind II
Q2. EcoRI is obtained from —
(a) Bacillus subtilis (b) Thermus aquaticus (c) Escherichia coli (d) Agrobacterium
Answer: (c) Escherichia coli
Q3. PCR was developed by —
(a) Boyer (b) Cohen (c) Mullis (d) Sanger
Answer: (c) Mullis
Q4. Taq polymerase is obtained from —
(a) Thermus aquaticus (b) Bacillus thuringiensis (c) E. coli (d) Saccharomyces
Answer: (a) Thermus aquaticus
Q5. Which is used as a vector in plant gene transfer?
(a) Lambda phage (b) pBR322 (c) Ti plasmid (d) Cosmid
Answer: (c) Ti plasmid
Q6. Sticky ends are produced by —
(a) DNA ligase (b) Restriction enzymes (c) Polymerase (d) Helicase
Answer: (b) Restriction enzymes
Q7. Which of the following is NOT a step of PCR?
(a) Denaturation (b) Annealing (c) Extension (d) Ligation
Answer: (d) Ligation
Q8. DNA in gel electrophoresis moves toward —
(a) Cathode (b) Anode (c) Both (d) Neither
Answer: (b) Anode
Q9. The selectable markers in pBR322 are —
(a) ampr and tetr (b) kanr and ampr (c) lac Z and tetr (d) ori and rop
Answer: (a) ampr and tetr
Q10. A bioreactor commonly used for cell culture is —
(a) Sparged stirred-tank (b) Centrifuge (c) Autoclave (d) Spectrophotometer
Answer: (a) Sparged stirred-tank
Fill in the Blanks
Q1. The full form of EFB is __________.
Answer: European Federation of Biotechnology
Q2. EcoRI recognises the palindromic sequence __________.
Answer: 5′-GAATTC-3′
Q3. The enzyme that joins DNA fragments is __________.
Answer: DNA ligase
Q4. Bacterial cells are made competent using __________.
Answer: calcium chloride (CaCl2)
Q5. The vector used for gene transfer in dicot plants is __________.
Answer: Ti plasmid of Agrobacterium tumefaciens
True / False
Q1. Restriction enzymes are also called molecular scissors.
Answer: True
Q2. Taq polymerase is heat sensitive.
Answer: False — Taq polymerase is thermostable.
Q3. Plasmids are extra-chromosomal, self-replicating DNA molecules.
Answer: True
Q4. DNA in gel electrophoresis is visualised under infrared light.
Answer: False — it is visualised under UV light.
Q5. Downstream processing includes purification and quality control.
Answer: True
Glossary
| Term | Meaning |
|---|---|
| Biotechnology | Use of living organisms or their parts to make useful products and services. |
| Genetic engineering | Manipulation and transfer of genes between organisms. |
| Bioprocess engineering | Sterile, large-scale culture of cells/microbes for production. |
| Restriction enzyme | Endonuclease that cuts DNA at specific palindromic sites. |
| Palindromic sequence | DNA sequence reading the same on both strands in 5’→3′ direction. |
| Sticky ends | Single-stranded overhangs produced after restriction cutting. |
| DNA ligase | Enzyme that seals nicks in DNA, joining fragments. |
| Vector | Carrier DNA molecule used to deliver foreign DNA to host. |
| Plasmid | Small circular extra-chromosomal DNA in bacteria, e.g., pBR322. |
| Selectable marker | Gene allowing identification of transformed cells (e.g., ampr). |
| Competent host | Cell made permeable to take up foreign DNA. |
| Gel electrophoresis | Technique to separate DNA fragments by size in agarose gel. |
| PCR | Polymerase Chain Reaction — exponential DNA amplification in vitro. |
| Taq polymerase | Heat-stable DNA polymerase from Thermus aquaticus. |
| Bioreactor | Large vessel for culturing cells under controlled conditions. |
| Downstream processing | Post-production steps: separation, purification, formulation, QC. |
| Transformation | Uptake of foreign DNA by a competent host cell. |
| Insertional inactivation | Loss of gene function due to insertion of foreign DNA into it. |
| Ti plasmid | Tumour-inducing plasmid of Agrobacterium used as plant vector. |
| Cosmid | Hybrid vector combining plasmid and lambda phage cos sites. |