Section 15 — Board Exam-Pattern Questions (Class 12 Biology · Chapter 9)
Chapter 9 (Biotechnology: Principles and Processes) is one of the most reliably scoring chapters in the Class 12 Biology Board paper. As a broad guide, about 5–8 marks of a typical paper tend to come from this chapter — spread across short recall questions, 2- and 3-mark process questions, and very often a 5-mark question on recombinant DNA technology, PCR or the selection of recombinants.
This section curates 24 high-yield Board questions in the format and weighting the examiners use:
| Marks | Number of Qs | Typical paper allocation |
|---|---|---|
| 1 mark | 7 questions (Q1–Q7) | 2–3 / paper |
| 2 marks | 6 questions (Q8–Q13) | 2 / paper |
| 3 marks | 6 questions (Q14–Q19) | 1–2 / paper |
| 5 marks | 5 questions (Q20–Q24) | 1 / paper (very high probability) |
| Total | 24 questions | ~24 marks worth |
How to use: Cover the answer, attempt it yourself scaled to the marks, then check against the model answer and its keyword notes.
No quiz at the end: Pair this set with the Solved Examples for a complete revision of the chapter.
What the Examiners Keep Asking
Most questions on Chapter 9 fall into six recurring themes:
1. Tools and principles (1- or 2-mark) — genetic vs bioprocess engineering, restriction enzymes, ligase, the role of ori and selectable markers.
2. Restriction enzymes and palindromes (2- or 3-mark) — naming convention, recognition sequence, sticky ends, why the same enzyme cuts both vector and gene.
3. Vectors and selection (2-, 3- or 5-mark) — features of a cloning vector, pBR322, insertional inactivation, the blue-white method, Ti plasmid and retroviruses.
4. Getting DNA into cells (2- or 3-mark) — competent cells, calcium and heat shock, micro-injection, biolistics.
5. PCR and DNA isolation (3- or 5-mark) — the three steps of a cycle, Taq polymerase, isolating pure DNA with lysozyme, protease and ethanol.
6. Downstream and scale-up (3- or 5-mark) — bioreactors, continuous culture, downstream processing and quality control.
1-Mark Questions (Q1–Q7)
Q1. [1 mark] Name the enzymes referred to as 'molecular scissors' in recombinant DNA technology.
Answer: Restriction endonucleases.
Keyword note: the full term, not just 'restriction enzymes', earns the mark cleanly.
Q2. [1 mark] From which bacterium is the thermostable Taq polymerase used in PCR obtained?
Answer: From Thermus aquaticus.
Q3. [1 mark] What is the property called by which any linked piece of DNA is made to replicate within a host cell?
Answer: It is due to the origin of replication (ori).
Q4. [1 mark] Name the divalent cation used to make bacterial cells competent to take up DNA.
Answer: Calcium (as calcium chloride, a divalent cation).
Q5. [1 mark · Assertion–Reason]
Assertion (A): DNA fragments move towards the anode during gel electrophoresis. Reason (R): DNA is a negatively charged molecule.
Options: (a) Both A and R true and R explains A. (b) Both true but R does not explain A. (c) A true, R false. (d) A false, R true.
Answer: (a) — DNA carries a net negative charge, so under an electric field it is drawn to the positive electrode (anode); R correctly explains A.
Q6. [1 mark] What is the technique of directly injecting recombinant DNA into the nucleus of an animal cell called?
Answer: Micro-injection.
Q7. [1 mark] Name the natural polymer, extracted from sea weeds, used as the matrix in gel electrophoresis.
Answer: Agarose.
2-Mark Questions (Q8–Q13)
Q8. [2 marks] Differentiate between exonuclease and endonuclease.
Answer: An exonuclease removes nucleotides from the ends of a DNA molecule, whereas an endonuclease makes cuts at specific positions within the DNA. Restriction enzymes used in genetic engineering are restriction endonucleases.
Keyword note: 'ends' versus 'within at specific positions' carries the two marks.
Q9. [2 marks] What is a palindromic sequence? Illustrate with the recognition site of EcoRI.
Answer: A palindromic sequence is one that reads the same on both strands when read in the same (5' to 3') direction. For EcoRI it is:
5' — GAATTC — 3' 3' — CTTAAG — 5'
Both strands read GAATTC in the 5' to 3' direction.
Q10. [2 marks] State any two features that a DNA sequence must have to serve as a cloning vector.
Answer: (i) An origin of replication (ori) so the linked DNA can replicate and its copy number is controlled; (ii) a selectable marker (such as an antibiotic-resistance gene) to identify and select transformants. A vector should also have single recognition sites for common restriction enzymes.
Q11. [2 marks] How are bacterial cells made competent, and how is recombinant DNA then forced into them?
Answer: Cells are treated with a divalent cation such as calcium, which increases DNA uptake through pores in the cell wall. The recombinant DNA is mixed with the cells on ice, given a brief heat shock at 42 degrees C, and returned to ice, enabling the cells to take up the DNA.
Q12. [2 marks] Name the plant and animal vectors used to deliver genes into eukaryotic cells, with their source organisms.
Answer: In plants, the Ti (tumour-inducing) plasmid of the bacterium Agrobacterium tumefaciens is used. In animals, disarmed retroviruses are used. Both are modified so that they are no longer pathogenic but can still deliver the gene of interest.
Q13. [2 marks] Why must the same restriction enzyme be used to cut both the vector and the foreign DNA?
Answer: The same enzyme produces the same kind of sticky ends on both pieces. These complementary single-stranded overhangs base-pair with each other, allowing DNA ligase to join them. If different enzymes were used, the ends would not match and no recombinant molecule could form.
3-Mark Questions (Q14–Q19)
Q14. [3 marks] Explain the three steps of one cycle of the Polymerase Chain Reaction.
Answer: (i) Denaturation — the double-stranded DNA is heated so the two strands separate. (ii) Annealing — two sets of primers, short oligonucleotides complementary to the flanking sequences, bind to the single strands. (iii) Extension — DNA polymerase (Taq) extends the primers using the supplied nucleotides and the template strand. Repeating the cycle amplifies the DNA about a billion-fold.
Keyword note: denaturation, annealing (primers), extension (Taq polymerase) — all three named.
Q15. [3 marks] Describe how a gene of interest is cloned into pBR322 at the tetracycline-resistance gene and how recombinants are then selected.
Answer: The foreign DNA is ligated at the BamHI site within the tetracycline-resistance gene, which inactivates that gene by insertional inactivation. The transformants are plated on ampicillin, where all plasmid-bearing cells grow, then transferred to tetracycline. Recombinants grow on ampicillin but not on tetracycline, while non-recombinants grow on both, which distinguishes the recombinant colonies.
Q16. [3 marks] Explain the blue-white selection method based on insertional inactivation of beta-galactosidase.
Answer: The foreign DNA is inserted within the coding sequence of the enzyme beta-galactosidase. If there is no insert, the intact enzyme acts on a chromogenic substrate and the colony turns blue. If an insert is present, the enzyme gene is inactivated, no colour is produced, and the colony remains white (colourless). The white colonies are the recombinants, identified on a single plate.
Q17. [3 marks] Outline the steps by which pure genomic DNA is isolated from a bacterial cell.
Answer: The cell wall is broken with lysozyme (cellulase for plant cells, chitinase for fungi) to release the contents. RNA is removed by ribonuclease and proteins by protease; other molecules are removed by suitable treatments. Adding chilled ethanol precipitates the purified DNA as fine threads that can be spooled out.
Q18. [3 marks] What is a bioreactor? Name any two systems that a stirred-tank bioreactor is fitted with, and state the function of the stirrer.
Answer: A bioreactor is a vessel in which raw materials are biologically converted into products using microbial, plant or animal cells under optimum conditions. A stirred-tank bioreactor has, among others, an oxygen delivery system, a foam control system, a temperature control system, a pH control system and sampling ports (any two). The stirrer (agitator) ensures even mixing and distributes oxygen throughout the vessel.
Q19. [3 marks] Explain how the restriction enzyme EcoRI is named, and state what a restriction endonuclease recognises before cutting.
Answer: The name is built as follows: E from the genus Escherichia, co from the species coli, R from the strain RY13, and the Roman numeral I for the order in which it was isolated from that strain. Before cutting, a restriction endonuclease inspects the DNA and binds to a specific palindromic recognition sequence, then cuts both strands of the backbone at defined points, leaving sticky ends.
5-Mark Questions (Q20–Q24)
Q20. [5 marks] Describe the steps of recombinant DNA technology in the correct sequence, from isolating the DNA to obtaining the final product.
Answer:
Recombinant DNA technology proceeds through a fixed sequence of steps.
1. Isolation of DNA: the cell is broken open with enzymes such as lysozyme, and RNA, protein and other molecules are removed so that pure DNA precipitates on adding chilled ethanol.
2. Cutting the DNA: purified DNA and the vector are cut with the same restriction endonuclease so that both carry matching sticky ends; progress is checked by gel electrophoresis.
3. Ligation into a vector: the gene of interest and the cut vector are mixed with DNA ligase, which seals the fragments into a recombinant molecule. The gene may first be amplified by PCR.
4. Transfer into the host: the recombinant DNA is introduced into a competent host (for example, E. coli made competent with calcium and a heat shock), where it replicates.
5. Culturing and product formation: the transformed cells are grown at large scale in a bioreactor so the gene is expressed, and the product is then recovered by downstream processing.
Keyword note: isolation, restriction digestion, ligation, transfer into competent host, culture and downstream processing — in order.
Q21. [5 marks] With the help of the cloning vector pBR322, explain the essential features of a cloning vector and the concept of insertional inactivation.
Answer:
A cloning vector must have certain features, well illustrated by pBR322.
Origin of replication (ori): the sequence from where replication starts; any DNA linked to it replicates within the host, and it also controls the copy number.
Selectable marker: pBR322 carries two antibiotic-resistance genes, ampR and tetR, which help select transformants because normal E. coli has no resistance to these antibiotics.
Cloning sites: a vector should have single recognition sites for common restriction enzymes; pBR322 has sites such as HindIII and EcoRI, BamHI and SalI (within the tetR gene), and PstI and PvuI (within the ampR gene).
Insertional inactivation: when foreign DNA is ligated at the BamHI site in the tetR gene, that gene is disrupted, so recombinants lose tetracycline resistance but keep ampicillin resistance. Plating on ampicillin then tetracycline distinguishes recombinants (ampicillin-resistant, tetracycline-sensitive) from non-recombinants (resistant to both). A simpler version uses insertional inactivation of beta-galactosidase, giving colourless recombinant colonies against blue non-recombinants.
Q22. [5 marks] What is the Polymerase Chain Reaction? Explain its three steps and the role of Taq polymerase, and state why the reaction is useful.
Answer:
PCR (Polymerase Chain Reaction) synthesises multiple copies of a gene or DNA segment of interest in vitro using two sets of primers and the enzyme DNA polymerase.
Three steps of each cycle: (i) Denaturation — heating separates the double-stranded DNA into single strands; (ii) Annealing — the primers, complementary to the flanking regions, bind to the single strands; (iii) Extension — DNA polymerase extends the primers using the supplied nucleotides and the template, forming new strands.
Role of Taq polymerase: the enzyme is a thermostable DNA polymerase from Thermus aquaticus. Because it stays active during the high-temperature denaturation step, the same enzyme can be reused through repeated cycles, so fresh enzyme is not needed each time.
Usefulness: repeated cycles amplify the segment about a billion times. The amplified DNA can be ligated into a vector for cloning, and PCR is also central to diagnosis and detecting very small amounts of DNA.
Q23. [5 marks] Explain how a foreign gene, once cloned, is expressed and its protein product obtained on an industrial scale.
Answer:
After the gene of interest is cloned, the aim in almost all recombinant technologies is to obtain a desirable protein, so the foreign gene must be expressed.
Expression: when a protein-coding gene is expressed in a foreign host, the product is called a recombinant protein. The cells carrying the cloned gene are first grown on a small scale and conditions are optimised to induce the protein.
Large-scale culture: small cultures cannot give useful quantities, so a continuous culture system is used, in which spent medium is drained out while fresh medium is added, keeping cells in the active log phase and yielding more biomass.
Bioreactors: to process large volumes (100 to 1000 litres), bioreactors are used. A stirred-tank bioreactor provides optimum temperature, pH, oxygen and nutrients, with an agitator, oxygen delivery, foam control, temperature and pH control and sampling ports.
Downstream processing: the product is then separated and purified, formulated with preservatives, put through clinical trials where relevant, and subjected to strict quality control before it is marketed.
Q24. [5 marks] Describe restriction enzymes under the following heads: discovery and naming, the nature of their recognition site, how they cut DNA, and their role in making recombinant DNA.
Answer:
Discovery and naming: the first restriction endonuclease, Hind II, was found to cut DNA only at a specific sequence of six base pairs. Over 900 restriction enzymes are now known. Naming follows a convention — genus, species, strain and a Roman numeral — for example, EcoRI is from Escherichia coli strain RY13.
Recognition site: each enzyme inspects the DNA and binds a specific palindromic sequence that reads the same on both strands in the 5' to 3' direction, such as GAATTC / CTTAAG.
How they cut: the enzyme cuts a little away from the centre of the palindrome, between the same two bases on opposite strands, leaving overhanging single-stranded sticky ends that can form hydrogen bonds with complementary ends.
Role in recombinant DNA: when the same enzyme cuts a vector and a source DNA, both carry matching sticky ends. These pair up and are sealed by DNA ligase, producing a recombinant molecule that combines DNA from different sources. This makes restriction enzymes the central cutting tool of genetic engineering.
End of Section 15
You have now worked through 24 high-yield Board questions spanning tools and principles, restriction enzymes and palindromes, cloning vectors and selection, competent cells, PCR, DNA isolation, bioreactors and downstream processing. If you can answer the five 5-markers (Q20–Q24) from memory with their key points, you have effectively secured this chapter's contribution to your paper.
Final tip: On exam day, draw and label diagrams wherever allowed — the action of EcoRI leaving sticky ends, the pBR322 map with ori and resistance genes, the PCR cycle, the stirred-tank bioreactor — examiners award marks for clear labelled structures. Tabulate comparison-type answers (exonuclease vs endonuclease, recombinant vs non-recombinant selection).