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Biotechnology: Principles and Processes

NCERT Class 12 · Biology Based on NCERT Class 12 Biology textbook · Free CBSE study kit

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Chapter Notes

Overview
This chapter introduces the fundamental principles and core techniques of modern biotechnology, focusing on genetic engineering and recombinant DNA technology. It explains how scientists can isolate, cut, join, and transfer DNA from one organism to another to produce useful products like insulin, vaccines, and enzymes. The one big idea a student must walk away with is that biotechnology relies on three key steps: cutting DNA with restriction enzymes, joining it with vectors using DNA ligase, and introducing the recombinant DNA into a host cell for replication and expression.
Key Concepts (24)
Biotechnology
The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.
e.g. Making curd, bread, or wine using microbes is a traditional form of biotechnology.
Genetic engineering
Techniques to alter the chemistry of genetic material (DNA and RNA) to introduce these into host organisms and thus change the phenotype of the host organism.
e.g. Inserting the human insulin gene into E. coli to produce insulin.
Bioprocess engineering
Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products.
e.g. Growing E. coli in a sterile bioreactor to produce recombinant proteins.
Recombinant DNA
A new combination of circular autonomously replicating DNA created in vitro by linking a gene of interest with a vector.
e.g. Linking the antibiotic resistance gene from Salmonella typhimurium with a plasmid vector to create the first recombinant DNA.
Restriction enzymes
Molecular scissors that cut DNA at specific recognition sequences, also called restriction endonucleases.
e.g. EcoRI cuts DNA at the palindromic sequence GAATTC.
Recognition sequence
A specific nucleotide sequence (usually 4-8 base pairs) that a restriction enzyme recognises and cuts at.
e.g. Hind II recognises a specific sequence of six base pairs.
Palindromic nucleotide sequence
A sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same (5' → 3' on one strand and 5' → 3' on the complementary strand).
e.g. 5' — GAATTC — 3' and 3' — CTTAAG — 5'.
Sticky ends
Single-stranded overhanging stretches left after restriction enzyme cuts, which form hydrogen bonds with complementary cut counterparts.
e.g. EcoRI leaves sticky ends that facilitate joining with DNA ligase.
DNA ligase
An enzyme that joins cut DNA molecules by sealing their ends.
e.g. Used to link the antibiotic resistance gene with the plasmid vector.
Cloning vectors
Plasmids or bacteriophages used to carry and replicate foreign DNA in a host cell.
e.g. pBR322 is a commonly used E. coli cloning vector.
Origin of replication (ori)
A sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells.
e.g. The ori sequence in pBR322 controls the copy number of the plasmid.
Selectable marker
A gene (e.g., antibiotic resistance) that helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants.
e.g. Ampicillin resistance gene (ampR) in pBR322.
Insertional inactivation
Inactivation of a gene (e.g., β-galactosidase) due to insertion of foreign DNA into its coding sequence, used to identify recombinants.
e.g. Insertion of DNA into the lacZ gene results in white colonies instead of blue on chromogenic substrate.
Competent host
A host cell (e.g., bacterial cell) treated to take up foreign DNA, usually by making it permeable using divalent cations like calcium.
e.g. E. coli cells treated with calcium chloride and given a heat shock become competent.
Transformation
A procedure through which a piece of DNA is introduced into a host bacterium.
e.g. Introducing recombinant plasmid into E. coli by heat shock method.
Polymerase Chain Reaction (PCR)
An in vitro technique to amplify a specific segment of DNA to billions of copies using primers and a thermostable DNA polymerase.
e.g. Amplifying the gene of interest from a small sample of DNA.
Thermostable DNA polymerase
A DNA polymerase isolated from Thermus aquaticus that remains active during high temperature denaturation of DNA.
e.g. Taq polymerase used in PCR.
Bioreactor
A vessel in which raw materials are biologically converted into specific products using microbial, plant, animal, or human cells under optimal conditions.
e.g. A stirred-tank bioreactor used to produce recombinant proteins in large volumes (100-1000 litres).
Downstream processing
The series of processes including separation and purification required to prepare a finished product after the biosynthetic stage.
e.g. Purifying insulin from bacterial culture and formulating it with preservatives.
Gel electrophoresis
A technique to separate DNA fragments based on their size by forcing them to move towards the anode through an agarose gel matrix under an electric field.
e.g. Separating restriction-digested DNA fragments to check the progression of digestion.
Elution
The step of cutting out and extracting separated DNA bands from the agarose gel.
e.g. Purifying the desired DNA fragment from the gel piece after electrophoresis.
T-DNA
A piece of DNA delivered by Agrobacterium tumifaciens to transform normal plant cells into tumor cells.
e.g. Ti plasmid modified into a cloning vector uses T-DNA to deliver genes of interest into plants.
Biolistics or gene gun
A method to introduce DNA into plant cells by bombarding them with high-velocity micro-particles of gold or tungsten coated with DNA.
e.g. Delivering a herbicide resistance gene into plant cells.
Micro-injection
A method where recombinant DNA is directly injected into the nucleus of an animal cell.
e.g. Introducing a gene into a mouse egg cell.
Formulas & Equations (2)
5' — GAATTC — 3' / 3' — CTTAAG — 5'
Reads as: Five prime G A A T T C three prime on one strand, and three prime C T T A A G five prime on the complementary strand.
Variables: G = Guanine, A = Adenine, T = Thymine, C = Cytosine. No SI units.
Use when: When identifying a palindromic recognition sequence for a restriction enzyme like EcoRI.
⚠ Students think palindrome means the same sequence read forward and backward on the same strand, but it means the same sequence read 5' to 3' on both strands.
Number of copies after n cycles of PCR = 2^n
Reads as: Number of copies equals two raised to the power n, where n is the number of cycles.
Variables: n = number of PCR cycles (dimensionless).
Use when: Calculating the amplification factor of a DNA segment after a given number of PCR cycles.
⚠ Students forget that the initial template is also amplified, so after 30 cycles, copies are approximately 2^30 (about 1 billion), not 2^30 minus 1.
How It Works
Construction of recombinant DNA using restriction enzymes and DNA ligase
  1. Step 1: Isolate the desired gene (e.g., antibiotic resistance gene) from source DNA by cutting with a specific restriction enzyme (e.g., EcoRI).
  2. Step 2: Cut the vector DNA (e.g., plasmid) with the same restriction enzyme to create complementary sticky ends.
  3. Step 3: Mix the cut gene and cut vector together; sticky ends form hydrogen bonds with complementary counterparts.
  4. Step 4: Add DNA ligase to seal the sugar-phosphate backbone, creating a recombinant DNA molecule.
  5. Step 5: Introduce the recombinant DNA into a host cell (e.g., E. coli) for replication and expression.
💡 The vector and source DNA must be cut with the same restriction enzyme to create compatible sticky ends; otherwise, recombinant DNA cannot be formed.
Polymerase Chain Reaction (PCR)
  1. Step 1: Denaturation — Heat the DNA to ~94-98°C to separate double-stranded DNA into single strands.
  2. Step 2: Primer annealing — Cool the mixture to ~50-65°C to allow two sets of primers (short oligonucleotides) to bind to complementary regions on the single-stranded DNA.
  3. Step 3: Extension — Raise temperature to ~72°C; thermostable DNA polymerase (Taq polymerase) extends the primers using nucleotides and the genomic DNA as template, synthesising new DNA strands.
  4. Step 4: Repeat steps 1-3 for 25-35 cycles to amplify the target DNA segment exponentially (approximately 2^n copies).
💡 The DNA polymerase used must be thermostable (from Thermus aquaticus) because it must remain active after the high-temperature denaturation step. Students often forget this key detail.
Making a bacterial host competent for transformation
  1. Step 1: Treat bacterial cells with a specific concentration of a divalent cation, such as calcium chloride.
  2. Step 2: Incubate the cells with recombinant DNA on ice.
  3. Step 3: Give a brief heat shock by placing the cells at 42°C.
  4. Step 4: Place the cells back on ice.
  5. Step 5: The bacteria take up the recombinant DNA through pores in the cell wall.
💡 The heat shock step is critical — students often miss the sequence: ice → 42°C → ice. Also, calcium ions increase the efficiency of DNA entry.
Gel electrophoresis for separation of DNA fragments
  1. Step 1: Prepare an agarose gel matrix (natural polymer from seaweeds).
  2. Step 2: Load DNA samples into wells at one end of the gel.
  3. Step 3: Apply an electric field — DNA fragments (negatively charged) move towards the anode (positive electrode).
  4. Step 4: Smaller fragments move faster and farther through the gel; larger fragments move slower.
  5. Step 5: Stain the gel with ethidium bromide and expose to UV radiation to visualise bright orange bands.
  6. Step 6: Cut out the desired band and extract DNA from the gel piece (elution).
💡 DNA is negatively charged due to phosphate groups, so it moves towards the anode. Students often incorrectly write that DNA moves towards the cathode.
Selection of recombinants using insertional inactivation
  1. Step 1: Insert foreign DNA into the coding sequence of the β-galactosidase gene (lacZ) in the vector.
  2. Step 2: Transform host cells with the recombinant vector.
  3. Step 3: Plate transformed cells on a medium containing a chromogenic substrate (e.g., X-gal).
  4. Step 4: Colonies with intact β-galactosidase (no insert) produce blue colour.
  5. Step 5: Colonies with insertional inactivation of β-galactosidase (recombinants) remain white (no colour).
💡 Blue colonies are non-recombinants; white colonies are recombinants. Students often reverse this.
Large-scale production in a bioreactor
  1. Step 1: Grow host cells (e.g., E. coli) harbouring the cloned gene in a small-scale culture in the laboratory.
  2. Step 2: Transfer the culture to a bioreactor (e.g., stirred-tank type) with large volume (100-1000 litres).
  3. Step 3: Provide optimal conditions: temperature, pH, substrate, salts, vitamins, and oxygen (via agitator or sparging).
  4. Step 4: Maintain sterile conditions; monitor and control foam, pH, and temperature.
  5. Step 5: Harvest the culture and extract the desired product (e.g., recombinant protein).
  6. Step 6: Subject the product to downstream processing: separation, purification, formulation with preservatives, and quality control.
💡 Bioreactors provide optimal growth conditions for large-scale production; they are not just for mixing — they control temperature, pH, oxygen, and foam.
Exam Traps — Don't Make These Mistakes
⚠ Confusing exonucleases and endonucleases
✗ Restriction enzymes are exonucleases that cut DNA from the ends.
→ Restriction enzymes are endonucleases — they cut at specific positions within the DNA, not from the ends. Exonucleases remove nucleotides from the ends.
✓ Restriction enzymes are endonucleases that make cuts at specific positions within the DNA at their recognition sequences.
⚠ Misunderstanding palindrome in DNA
✗ A palindromic DNA sequence reads the same forward and backward on the same strand, like 'MALAYALAM'.
→ In DNA, palindrome means the sequence reads the same on the two strands when orientation of reading is kept the same (5' → 3' on one strand and 5' → 3' on the complementary strand). It is not the same as word palindromes.
✓ A palindromic DNA sequence is one where the sequence on one strand (5' → 3') is identical to the sequence on the complementary strand (5' → 3'), e.g., 5' GAATTC 3' and 3' CTTAAG 5'.
⚠ Direction of DNA movement in gel electrophoresis
✗ DNA moves towards the cathode (negative electrode) because it is negatively charged.
→ Opposites attract: negatively charged DNA moves towards the positive electrode (anode), not the cathode.
✓ DNA is negatively charged and moves towards the anode (positive electrode) during gel electrophoresis.
⚠ Confusing selectable marker with cloning site
✗ The selectable marker is the restriction site where foreign DNA is inserted.
→ The selectable marker is an antibiotic resistance gene used to identify transformants. The cloning site is the restriction site where foreign DNA is inserted, often within a selectable marker for insertional inactivation.
✓ The selectable marker (e.g., ampR) helps identify transformants, while the cloning site (e.g., BamH I site) is where foreign DNA is ligated.
⚠ Forgetting the role of ori in copy number
✗ The origin of replication (ori) is only for starting replication.
→ The ori sequence also controls the copy number of the linked DNA. A vector with a high-copy-number ori allows many copies of the target DNA to be recovered.
✓ The ori sequence is responsible for initiating replication and also controls the copy number of the linked DNA in the host cell.
⚠ Mixing up the steps of PCR
✗ PCR steps are: annealing, denaturation, extension.
→ The correct order is denaturation first (to separate strands), then annealing (primers bind), then extension (DNA synthesis).
✓ The three steps of PCR in order are: (i) Denaturation, (ii) Primer annealing, (iii) Extension of primers.
Compare & Contrast
Site of action on DNA
Exonuclease
Endonuclease
Exonucleases remove nucleotides from the ends of DNA, while endonucleases make cuts at specific positions within the DNA.
Location and replication
Plasmid DNA
Chromosomal DNA
Plasmid DNA is extra-chromosomal, circular, and replicates independently of the chromosomal DNA; chromosomal DNA is the main coding strand and replicates as part of the chromosome.
Scale and control
Stirred-tank bioreactor
Shake flask
A stirred-tank bioreactor provides optimal conditions (temperature, pH, oxygen, mixing) for large volumes (100-1000 litres), while a shake flask is used for small-scale cultures with limited control.
Exam Focus
Marks pattern: 2 MCQs (1 mark each), 1 Very Short Answer (1 mark), 1 Short Answer (3 marks), 1 Long Answer (5 marks) — total approximately 10-12 marks
Most asked: Restriction enzymes: recognition sequences, palindromic sequences, sticky ends · Cloning vectors: features (ori, selectable marker, cloning sites), pBR322 · PCR: steps, thermostable DNA polymerase, amplification · Competent host preparation and transformation · Gel electrophoresis: principle and procedure · Insertional inactivation and blue-white selection
Don't skip: Describe the steps in recombinant DNA technology with a diagram (5 marks); Explain the role of restriction enzymes and DNA ligase in forming recombinant DNA (3 marks); Describe the process of PCR and its applications (3 marks); Explain the features of a cloning vector with reference to pBR322 (3 marks)
One-Line Revision (read aloud before the exam)
  1. Biotechnology integrates natural science and organisms for products and services.
  2. Genetic engineering alters DNA/RNA to change the phenotype of host organisms.
  3. Bioprocess engineering maintains sterile conditions for large-scale microbial growth.
  4. Recombinant DNA is created by linking a gene of interest with a vector using DNA ligase.
  5. Restriction enzymes are molecular scissors that cut DNA at specific palindromic recognition sequences.
  6. Sticky ends are single-stranded overhangs that facilitate joining of DNA fragments.
  7. DNA ligase seals the sugar-phosphate backbone to form recombinant DNA.
  8. A cloning vector must have an origin of replication (ori), a selectable marker, and unique restriction sites.
  9. pBR322 is a common E. coli vector with ampR and tetR selectable markers.
  10. Insertional inactivation of β-galactosidase gene allows blue-white selection of recombinants.
  11. PCR amplifies DNA using denaturation, primer annealing, and extension with thermostable DNA polymerase.
  12. Taq polymerase from Thermus aquaticus is used in PCR because it withstands high temperatures.
  13. Competent host cells are made by treating with calcium chloride and giving a heat shock.
  14. Gel electrophoresis separates DNA fragments by size; DNA moves towards the anode.
  15. Ethidium bromide staining and UV light are used to visualise DNA bands in gel.
  16. Bioreactors provide optimal conditions (temperature, pH, oxygen) for large-scale production.
  17. Downstream processing includes separation, purification, formulation, and quality control of the product.
  18. Agrobacterium tumifaciens delivers T-DNA into plants; used as a vector for plant genetic engineering.
  19. Retroviruses can deliver genes into animal cells and are used as vectors after being disarmed.
  20. The first recombinant DNA was constructed by linking an antibiotic resistance gene with a plasmid in 1972 by Cohen and Boyer.

MCQs — 10 Questions with Answers

Q1. Restriction enzymes discovered by Herbert Boyer have the ability to cut DNA at specific locations and produce which of the following?

  • A. Blunt ends that cannot be joined to other DNA
  • B. Sticky ends that enable precise joining of DNA fragments ✓
  • C. Circular plasmid molecules
  • D. Multiple copies of the entire genome

Answer: B — Restriction enzymes create sticky ends on DNA strands, which are complementary single-stranded overhangs that allow precise joining of DNA fragments from different sources.

Q2. A plasmid is described as an ideal vector for recombinant DNA technology because it:

  • A. Is part of the bacterial chromosome and replicates with it
  • B. Is a small, circular, autonomously replicating DNA that exists independently in bacterial cytoplasm ✓
  • C. Cannot carry alien DNA due to its small size
  • D. Requires integration with the origin of replication to function

Answer: B — Plasmids are autonomously replicating circular DNAs that float freely in bacterial cytoplasm, making them ideal vectors as they can carry and independently replicate alien DNA inserted into them.

Q3. Why is the origin of replication essential for the multiplication of alien DNA in a host organism?

  • A. It provides the enzyme DNA polymerase to the host cell
  • B. It is a specific DNA sequence that initiates replication, allowing alien DNA to be copied and inherited in progeny cells ✓
  • C. It prevents the alien DNA from being degraded by restriction enzymes
  • D. It allows the alien DNA to bypass the cell membrane

Answer: B — The origin of replication is a specific DNA sequence responsible for initiating replication; when alien DNA is linked to it, the alien DNA becomes part of a chromosome and replicates along with host DNA.

Q4. The enzyme DNA ligase plays which role in the construction of recombinant DNA?

  • A. It cuts DNA at specific sequences to produce fragments
  • B. It joins the cut ends of DNA molecules, linking alien DNA to plasmid vectors ✓
  • C. It replicates the plasmid DNA in the host organism
  • D. It removes unwanted genes from the alien DNA

Answer: B — DNA ligase acts on cut DNA molecules and joins their ends, enabling the linking of an alien DNA fragment to a plasmid vector to create recombinant DNA.

Q5. In the historic 1972 experiment by Cohen and Boyer, the antibiotic resistance gene was transferred into E. coli using a plasmid vector from Salmonella typhimurium. What was the primary advantage of this approach over traditional hybridisation?

  • A. Only the desired antibiotic resistance gene was transferred without undesirable genes ✓
  • B. E. coli could reproduce sexually like higher organisms
  • C. The plasmid integrated directly with the bacterial chromosome
  • D. Multiple undesirable genes were eliminated automatically

Answer: A — Genetic engineering via restriction enzymes and plasmids allows transfer of only the desired gene without undesirable genes, overcoming the limitation of traditional breeding where unwanted genes are multiplied along with desired ones.

Q6. A piece of alien DNA is introduced into a bacterial cell but fails to replicate in the progeny. Which of the following is the most likely reason?

  • A. The alien DNA was cut by restriction enzymes into fragments
  • B. The alien DNA did not integrate into the chromosome and lacks an origin of replication ✓
  • C. The bacterial cell has no DNA ligase enzyme
  • D. The plasmid vector was too large for the bacterial cell

Answer: B — Alien DNA must integrate into the host chromosome, which contains an origin of replication; without integration and this sequence, the alien DNA cannot replicate and is lost in progeny cells.

Q7. Which of the following statements is INCORRECT regarding the principles of biotechnology?

  • A. Genetic engineering introduces desirable genes without undesirable genes into target organisms
  • B. Bioprocess engineering maintains sterile conditions to allow growth of only desired microbes
  • C. Asexual reproduction permits more genetic variation than genetic engineering techniques ✓
  • D. Recombinant DNA is created by linking alien DNA to plasmids using DNA ligase

Answer: C — Asexual reproduction preserves genetic information without variation, whereas sexual reproduction and especially genetic engineering enable genetic variation and new combinations; the statement incorrectly claims asexual reproduction provides more variation.

Q8. Consider two statements: (Assertion) Sticky ends produced by restriction enzymes are crucial for recombinant DNA formation. (Reason) Sticky ends are complementary single-stranded overhangs that allow precise joining of DNA fragments from different sources. Which of the following is correct?

  • A. Both assertion and reason are true, and reason explains the assertion ✓
  • B. Both assertion and reason are true, but reason does not explain the assertion
  • C. Assertion is true, but reason is false
  • D. Both assertion and reason are false

Answer: A — Sticky ends are indeed crucial for recombinant DNA formation because their complementary nature enables precise and specific joining of DNA fragments, making the reason a correct explanation of the assertion.

Q9. If a plasmid vector has a molecular weight of 5 kb (kilobases) and an alien DNA fragment is 8 kb, the resulting recombinant DNA molecule would have a molecular weight of approximately:

  • A. 3 kb (difference)
  • B. 5 kb (plasmid weight only)
  • C. 13 kb (sum of both) ✓
  • D. 40 kb (product of both)

Answer: C — When an alien DNA fragment (8 kb) is joined to a plasmid vector (5 kb) using DNA ligase, the total molecular weight of the resulting recombinant DNA is the sum: 5 + 8 = 13 kb.

Q10. The European Federation of Biotechnology (EFB) definition of biotechnology as 'integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services' encompasses both traditional and modern biotechnology. Why is making curd using Lactobacillus considered biotechnology under this definition, even though it predates modern genetic engineering?

  • A. Because curd production uses genetically modified organisms exclusively
  • B. Because it involves using living organisms (Lactobacillus cells) to produce a useful product (curd) through integration of natural science ✓
  • C. Because curd production requires restriction enzymes and plasmids
  • D. Because the EFB definition only applies to modern molecular biotechnology

Answer: B — The EFB definition is broad and inclusive; making curd uses living Lactobacillus organisms to produce a useful product through fermentation, fitting the definition of integrating organisms for products and services, making it biotechnology even without genetic modification.

Flashcards

What are restriction enzymes and what is their key property discovered by Boyer?

Restriction enzymes are 'molecular scissors' that cut DNA at specific locations and create sticky ends, allowing precise DNA fragments to be joined together.

Define recombinant DNA and state when it was first created.

Recombinant DNA is a new combination of circular autonomously replicating DNA created in vitro by joining an alien DNA fragment to a plasmid vector, first accomplished by Cohen and Boyer in 1972.

What is the role of the origin of replication in genetic engineering?

The origin of replication is a specific DNA sequence in chromosomes that initiates replication, allowing alien DNA linked to it to be copied and inherited in the host organism.

What is the function of DNA ligase in constructing recombinant DNA?

DNA ligase is an enzyme that joins the cut ends of DNA molecules together, making it possible to link an alien DNA fragment to a plasmid vector.

Why is a plasmid considered a suitable vector for transferring alien DNA?

A plasmid is a suitable vector because it is a small, autonomously replicating circular DNA that can carry alien genes into a host cell and replicate independently from chromosomal DNA.

What is the difference between genetic engineering and bioprocess engineering?

Genetic engineering alters DNA chemistry to change an organism's phenotype, while bioprocess engineering maintains sterile conditions to grow desired microbes or cells in large quantities for product manufacture.

How does genetic engineering overcome the limitation of traditional hybridisation breeding?

Genetic engineering allows isolation and transfer of only desired genes without undesirable genes, whereas traditional hybridisation often multiplies undesirable genes along with desired ones.

What is cloning in the context of biotechnology?

Cloning is the process of making multiple identical copies of any template DNA so it can be replicated and inherited in the host organism.

What does the European Federation of Biotechnology (EFB) define biotechnology as?

The EFB defines biotechnology as the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

Why must alien DNA inserted into a host organism become integrated into its genome?

Alien DNA must integrate into the genome so it becomes part of a chromosome with an origin of replication, enabling it to replicate and be inherited in progeny cells.

Important Board Questions

Define recombinant DNA and state the year in which the first recombinant DNA was constructed. Name the two scientists responsible for this achievement. [2 marks]

Recombinant DNA is a new circular DNA created in vitro by joining alien DNA to a plasmid vector; constructed in 1972 by Cohen and Boyer using restriction enzymes and DNA ligase.

Explain why origin of replication is essential for the multiplication and inheritance of alien DNA in a host organism. Describe the role of each component in creating recombinant DNA: restriction enzymes, plasmid vectors, and DNA ligase. [5 marks]

Origin of replication initiates DNA copying and allows alien DNA integrated into chromosomes to replicate; restriction enzymes cut DNA at specific sites creating sticky ends; plasmid vectors carry alien DNA; DNA ligase joins cut DNA ends. Show how these work together: cutting → linking to plasmid → integration → replication.

Genetic engineering overcomes the limitations of traditional hybridisation in plant and animal breeding. Explain how this is achieved by describing the three basic steps of genetic modification and compare the outcomes with traditional breeding. Include the significance of being able to isolate and transfer only desired genes without undesirable genes. [6 marks]

Three steps: (1) isolation of desired genes using restriction enzymes, (2) cloning via plasmid vectors and DNA ligase creating recombinant DNA, (3) transfer and expression in host organism. Traditional breeding multiplies undesirable genes along with desired; genetic engineering transfers only target genes via origin of replication integration, providing precise genetic combinations and phenotypic changes without genetic load.

Assertion-Reason Questions for Biotechnology: Principles and Processes

Q1. Assertion (A):

Restriction enzymes are called molecular scissors because they cut DNA at specific recognition sequences.

Reason (R):

Restriction enzymes recognize and cut only at specific base-pair sequences, producing predictable fragments with sticky ends.

Answer: A — Both A and R are true, and R is the correct explanation of A — Both the assertion and reason are true. The reason directly explains why restriction enzymes are called molecular scissors—their specificity and ability to cut DNA precisely at defined sequences. This is the core reason for their nickname.

Q2. Assertion (A):

Plasmids cannot be used as vectors in genetic engineering if they lack an origin of replication.

Reason (R):

Without an origin of replication, the foreign DNA inserted into the plasmid will not replicate in the host cell.

Answer: A — Both A and R are true, and R is the correct explanation of A — Both statements are true, and the reason correctly explains the assertion. The origin of replication is essential for autonomous replication of the plasmid and the linked foreign DNA in the host organism.

Q3. Assertion (A):

DNA ligase is essential for forming recombinant DNA, whereas DNA polymerase is not needed in the initial construction of recombinant DNA.

Reason (R):

DNA ligase joins the ends of cut DNA, while DNA polymerase synthesizes new DNA strands; only ligase is involved in the joining step of recombination.

Answer: B — Both A and R are true, but R is NOT the correct explanation of A — Both statements are true, but the reason does not fully explain the assertion. While DNA polymerase is not used in the initial construction/joining step, it becomes essential later for replicating the recombinant DNA in the host organism. The reason is only partially explanatory.

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Case-Based Questions (CBQ) for Biotechnology: Principles and Processes

Case Study:

A biotech company in India aims to produce recombinant insulin using E. coli as a biofactory. The team isolates a human insulin gene (hINS) and uses the plasmid pBR322 (which contains an origin of replication, an ampicillin resistance marker, and EcoRI restriction sites) as the vector. They cut both the insulin gene and the plasmid with EcoRI restriction enzyme and use DNA ligase to join them. The recombinant plasmid is then introduced into competent E. coli cells. After selection on ampicillin-containing plates, colonies are grown in fermentation tanks under sterile, temperature-controlled conditions for large-scale insulin production.

Q1. Why is the pBR322 plasmid suitable as a vector for this recombinant insulin production?

  • A. It contains an origin of replication, antibiotic resistance marker, and EcoRI restriction sites for gene insertion ✓
  • B. It is the only plasmid that can carry eukaryotic genes
  • C. It produces insulin naturally without genetic modification
  • D. It prevents bacterial contamination in fermentation tanks

Answer: A

Q2. What is the role of the ampicillin resistance marker in the pBR322 plasmid during this experiment?

  • A. It helps identify and select only the E. coli cells that have taken up the recombinant plasmid ✓
  • B. It prevents all bacterial contamination in the growth medium
  • C. It ensures that the insulin gene is expressed at high levels
  • D. It increases the replication rate of the plasmid in E. coli

Answer: A

Q3. For the recombinant insulin to be produced in large quantities, which of the following is NOT directly necessary?

  • A. Sterile conditions and temperature control in fermentation tanks
  • B. A prokaryotic promoter sequence upstream of the insulin gene
  • C. An origin of replication in the plasmid vector
  • D. Daily replacement of the fermentation medium ✓

Answer: D

Q4. If the insulin gene lacks a proper prokaryotic ribosome binding site (Shine-Dalgarno sequence), the MOST LIKELY outcome would be:

  • A. The insulin gene would not be transcribed into mRNA in E. coli
  • B. The mRNA would be produced but not efficiently translated into insulin protein ✓
  • C. The plasmid would not replicate in E. coli cells
  • D. The E. coli cells would reject the recombinant plasmid entirely

Answer: B

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Previous Year Questions (PYQ) for Biotechnology: Principles and Processes

Real CBSE board exam questions from past papers — free to attempt.

CBSE 2026 · Set 57/1/1 · 1 mark

Pomato was produced by fusing protoplasts of

  • Tomato and Potato
  • Pomegranate and Tomato
  • Pomegranate and Potato
  • Pomegranate, Potato and Tomato

CBSE 2026 · Set 57/1/1 · 1 mark

Match Column-I with Column-II and choose the correct option: Column-I: a. Biolistic gun, b. Chitinase, c. Ti, d. Ca++ | Column-II: i. Bacterial cell, ii. Tumour inducing, iii. Animal cell, iv. Fungal cell, v. Plant cell

  • i ii iii iv
  • ii v i iii
  • v iv ii i
  • v i iv ii

CBSE 2026 · Set 57/1/1 · 3 marks

Observe the given picture carefully. A mixture of DNA with fragments ranging from 100 base pairs to 1800 base pairs were separated by electrophoresis on agarose gel with the following arrangement: (a) What result will be obtained in staining with ethidium bromide? Explain with reasons. (1 mark) (b) The above setup was modified as shown below and a band with 100 base pairs was obtained at X. What changes were made to the previous design to get a band at 'X'. Why did the band appear at 'X'? (2 marks)

CBSE 2026 · Set 57/1/2 · 1 mark

Match Column-I with Column-II and choose the correct option: a. Biolistic gun, b. Chitinase, c. Ti, d. Ca++ with i. Bacterial cell, ii. Tumour inducing, iii. Animal cell, iv. Fungal cell, v. Plant cell

  • a-i, b-ii, c-iii, d-iv
  • a-ii, b-v, c-i, d-iii
  • a-v, b-iv, c-ii, d-i
  • a-v, b-i, c-iv, d-ii

CBSE 2026 · Set 57/1/2 · 1 mark

Which one of the following is not a component required for Polymerase Chain Reaction (PCR)?

  • Template DNA
  • Primers
  • DNA Ligase
  • DNA Polymerase

CBSE 2026 · Set 57/1/2 · 2 marks

Write the importance of following in biotechnology: (a) Ori (Origin of Replication) (1 mark) (b) Enzyme Polymerase used in Polymerase Chain Reaction (1 mark)

CBSE 2026 · Set 57/1/3 · 1 mark

Which of the following statements about plasmids is incorrect?

  • Plasmids have the ability to replicate within the bacterial cell.
  • Their replication is controlled by chromosomal DNA.
  • They are autonomously replicating circular extra-chromosomal DNA.
  • They often carry antibiotic resistant genes.

CBSE 2026 · Set 57/1/3 · 1 mark

Expression of a particular gene helps to identify transformants. What is this gene known as?

  • Plasmid
  • Structural gene
  • Selectable marker
  • Intron

CBSE 2026 · Set 57/1/3 · 3 marks

Observe the given picture carefully. A mixture of DNA with fragments ranging from 100 base pairs to 1800 base pairs were separated by electrophoresis on agarose gel with the following arrangement: (a) What result will be obtained in staining with ethidium bromide? Explain with reasons. (b) The above setup was modified as shown below and a band with 100 base pairs was obtained at X. What changes were made to the previous design to get a band at 'X'. Why did the band appear at 'X'?

CBSE 2026 · Set 57/2/1 · 1 mark

Which of the following is used to visualize DNA bands under UV light after gel electrophoresis ?

  • Acetocarmine
  • Safranine
  • Ethidium Bromide
  • Potassium Iodide

CBSE 2026 · Set 57/2/1 · 1 mark

Assertion (A) : DNA Ligase is used to join DNA fragments. Reason (R) : It catalyses the formation of glycosidic bonds between nucleotides.

  • Both Assertion (A) and Reason (R) are true and Reason (R) is the correct explanation for Assertion (A).
  • Both Assertion (A) and Reason (R) are true, but Reason (R) is not correct explanation for Assertion (A).
  • Assertion (A) is true, Reason (R) is false.
  • Assertion (A) is false, Reason (R) is true.

CBSE 2026 · Set 57/2/1 · 2 marks

How do scientists use microinjection and gene gun in biotechnology ?

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