Section 14 — Solved Examples
This is the dedicated problem set for Chapter 9 (Biotechnology: Principles and Processes). The 30 worked examples below are arranged in three tiers — concept checks (definitions, enzyme naming, the meaning of key terms), application and scenarios (case-style problems on cutting and joining DNA, vectors, transformation and PCR), and analytical and multi-concept (reasoning problems that stitch the whole recombinant-DNA workflow together, from an intact cell to a purified protein).
Most of these questions ask you to explain, predict and justify rather than merely name. In this chapter, naming a tool is rarely enough — you are usually asked why the same restriction enzyme cuts both the vector and the gene, how a recombinant colony is told apart from a non-recombinant one, or what keeps the Taq polymerase working when everything else would fall apart.
How to use this section
- Concept Checks (Q1–Q10): Quick recall of principles, enzymes, palindromes and vector features. If more than one or two trip you up, revisit Sections 2–8.
- Application and Scenarios (Q11–Q20): The workhorse 2- and 3-mark problems — pBR322 selection, plant and animal vectors, competent cells, DNA isolation, the PCR cycle and the bioreactor.
- Analytical and Multi-Concept (Q21–Q30): Multi-step reasoning that ties several ideas together, the kind that separates a passable answer from a full-mark one.
Total target time: around an hour for a complete revision sweep.
Note: This section is for practice and revision only — there is no quiz at the end. Treat each answer as a model of the detail an examiner expects.
Memory Capsule — Facts Worth Locking In
Before working through the problems, fix these ten high-yield facts firmly in mind:
| # | Fact | Where it is tested |
|---|---|---|
| 1 | Two core techniques of modern biotechnology — genetic engineering and bioprocess engineering (sterile large-scale growth) | Principles |
| 2 | Cohen and Boyer built the first recombinant DNA in 1972, linking an antibiotic-resistance gene to a Salmonella plasmid | History and principle |
| 3 | Hind II was the first restriction endonuclease; it recognises a specific six base-pair sequence | Restriction enzymes |
| 4 | Enzyme name — genus + species letters + strain + Roman numeral, e.g. EcoRI from Escherichia coli RY13 | Naming convention |
| 5 | A DNA palindrome reads the same 5' to 3' on both strands, e.g. GAATTC / CTTAAG; cuts leave sticky ends | Palindromes |
| 6 | In gel electrophoresis DNA (negatively charged) moves to the anode; smaller fragments travel farther; stained with ethidium bromide under UV | Electrophoresis |
| 7 | A cloning vector needs ori, a selectable marker and single cloning sites; pBR322 carries ampR and tetR | Vectors |
| 8 | Insertional inactivation — insert at the BamHI site knocks out tetracycline resistance; the blue-white test uses beta-galactosidase | Selection |
| 9 | Plant vector — Ti plasmid of Agrobacterium tumefaciens; animal vector — disarmed retroviruses | Eukaryotic vectors |
| 10 | PCR — denaturation, annealing, extension; uses Taq polymerase from Thermus aquaticus; competent cells made with CaCl2 and a 42 degree C heat shock | PCR and transformation |
Pro tip: When a question involves selecting a recombinant, always mention both halves of the logic — the marker that confirms the cell took up the vector, and the second marker (or colour) that is switched off by the insert.
Concept Checks (Q1–Q10)
Q1. Name the two core techniques that enabled the birth of modern biotechnology.
Answer: The two techniques are genetic engineering and bioprocess engineering. Genetic engineering alters the chemistry of the genetic material (DNA and RNA) and introduces it into a host to change the host's phenotype. Bioprocess engineering maintains a sterile, contamination-free environment so that only the desired microbe or cell grows in large quantities to manufacture products such as antibiotics, vaccines and enzymes.
Q2. List the three basic steps involved in genetically modifying an organism.
Answer: (i) Identification of the DNA carrying the desirable gene; (ii) introduction of that DNA into the host; and (iii) maintenance of the introduced DNA in the host and its transfer to the progeny. Only when the alien DNA becomes part of a replicating chromosome or vector can it multiply and be inherited.
Q3. What is the origin of replication, and why is it essential for cloning?
Answer: The origin of replication (ori) is a specific DNA sequence from where replication begins. Any piece of DNA linked to it can be made to replicate inside a host cell. It is essential because an alien piece of DNA on its own cannot multiply; only when it is joined to a sequence carrying an ori does it copy itself along with the host. The ori also controls the copy number of the linked DNA.
Q4. Explain how the restriction enzyme EcoRI gets its name.
Answer: The first letter comes from the genus (E for Escherichia), the next two from the species (co for coli), the next letter from the strain (R from RY13), and the Roman numeral (I) shows the order in which it was isolated from that strain. So EcoRI is the first restriction enzyme isolated from Escherichia coli strain RY13.
Q5. Distinguish between an exonuclease and an endonuclease.
Answer: Both are nucleases. An exonuclease removes nucleotides from the ends of a DNA molecule. An endonuclease makes cuts at specific positions within the DNA. Restriction enzymes used in genetic engineering are restriction endonucleases, because they cut internally at defined recognition sequences.
Q6. What is a palindromic sequence in DNA? Give one example.
Answer: A DNA palindrome is a sequence of base pairs that reads the same on the two strands when the direction of reading is kept the same (5' to 3'). For example:
5' — GAATTC — 3' 3' — CTTAAG — 5'
Reading either strand in the 5' to 3' direction gives GAATTC. Restriction endonucleases recognise such palindromic sequences.
Q7. What are sticky ends, and why are they called so?
Answer: When a restriction enzyme cuts DNA a little away from the centre of the palindrome, but between the same two bases on opposite strands, it leaves short single-stranded overhangs at each end. These are the sticky ends. They are called so because they can form hydrogen bonds with complementary overhangs on other DNA fragments cut by the same enzyme, and this stickiness helps DNA ligase join the fragments.
Q8. In gel electrophoresis, in which direction do DNA fragments move, and why? Name the matrix commonly used.
Answer: DNA fragments move towards the anode (the positive electrode) because DNA is a negatively charged molecule. The matrix commonly used is agarose, a natural polymer extracted from sea weeds. The gel acts as a sieve, so the smaller a fragment, the farther it moves.
Q9. How are separated DNA fragments made visible on an agarose gel?
Answer: Pure DNA cannot be seen in visible light. The gel is stained with ethidium bromide and then exposed to UV radiation, which makes the DNA show up as bright orange bands. The band of interest can then be cut out from the gel and recovered — a step called elution.
Q10. Name any three antibiotic-resistance genes used as selectable markers in E. coli, and state why they work as markers.
Answer: Genes conferring resistance to ampicillin, tetracycline, chloramphenicol or kanamycin are commonly used. They work because normal E. coli cells carry no resistance to these antibiotics. So only cells that have taken up the vector (and hence its resistance gene) survive on the antibiotic-containing medium, allowing transformants to be selected out from non-transformants.
Application and Scenarios (Q11–Q20)
Q11. A student cuts the vector DNA with EcoRI and the source DNA with BamHI, then tries to join them. The recombinant does not form. Explain why.
Answer: The two enzymes leave different sticky ends. For the overhangs to base-pair and be sealed by DNA ligase, the fragments must have complementary single-stranded ends, and that happens only when the same restriction enzyme cuts both the vector and the source DNA. Cutting one piece with EcoRI and the other with BamHI produces incompatible ends, so no recombinant molecule can form.
Q12. A foreign gene is ligated into the BamHI site of the tetracycline-resistance gene of pBR322. Explain, step by step, how recombinant colonies are then selected.
Answer: Inserting the foreign DNA at the BamHI site disrupts the tetracycline-resistance gene, so recombinants lose tetracycline resistance but keep ampicillin resistance. The transformants are first plated on ampicillin, where all cells carrying the plasmid grow. These are then transferred to a tetracycline medium. Recombinants grow on ampicillin but not on tetracycline, while non-recombinants grow on both. This difference identifies the recombinant colonies. Here one resistance gene selects the transformants and the other, by being inactivated, selects the recombinants.
Q13. Selecting recombinants by two antibiotic plates is described as cumbersome. Describe the alternative method based on colour.
Answer: The alternative uses insertional inactivation of the enzyme beta-galactosidase. The foreign DNA is inserted within the coding sequence of this enzyme. If there is no insert, the intact enzyme acts on a chromogenic substrate to give blue colonies. If there is an insert, the enzyme gene is inactivated, no colour is produced, and the colonies are white (colourless) — these are the recombinants. A single plate identifies recombinants, so it is far simpler than double-antibiotic plating.
Q14. To transfer a gene into a dicot plant, which natural vector is modified, and what makes it suitable?
Answer: The tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens is modified into a cloning vector. In nature this pathogen delivers a piece of DNA called T-DNA into plant cells and turns them into a tumour. The plasmid is disarmed — made non-pathogenic — while retaining its natural ability to transfer DNA, so it can carry a gene of interest into a variety of plants without causing disease.
Q15. How are genes delivered into animal cells using a natural biological vector, and what precaution is taken?
Answer: Retroviruses are used. In nature they can transform normal animal cells into cancerous cells by delivering their genetic material. For genetic engineering they are disarmed so they can no longer cause disease, but they retain the ability to insert a desirable gene into the animal cell. Once ligated into such a vector, the gene is transferred into the animal host, where it multiplies.
Q16. Bacterial cells will not normally take up a plasmid. Describe how they are made competent and how the recombinant DNA is then forced in.
Answer: Because DNA is hydrophilic, it cannot pass through the cell membrane on its own. The cells are made competent by treating them with a divalent cation, usually calcium (CaCl2), which increases the efficiency with which DNA passes through pores in the cell wall. The recombinant DNA is then mixed with the cells on ice, given a brief heat shock at 42 degrees C, and returned to ice. This sudden temperature change helps the bacteria take up the recombinant DNA.
Q17. Apart from making cells competent, name and briefly describe two other methods of introducing alien DNA into a host cell.
Answer: (i) Micro-injection — recombinant DNA is injected directly into the nucleus of an animal cell using a fine needle. (ii) Biolistics (gene gun) — suitable for plants; cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA. A third route uses disarmed pathogen vectors that carry the DNA in when they infect the cell.
Q18. Outline how genomic DNA is isolated in pure form from bacterial cells before it can be cut with restriction enzymes.
Answer: The cell is first broken open with a suitable enzyme — lysozyme for bacteria (cellulase for plant cells, chitinase for fungi) — to release its contents. The unwanted molecules are then removed: RNA by ribonuclease and proteins by protease; other molecules are removed by appropriate treatments. Finally, adding chilled ethanol precipitates the purified DNA, which appears as fine collectable threads that can be spooled out of the suspension.
Q19. Name the three steps of one PCR cycle and state what happens in each.
Answer: (i) Denaturation — the double-stranded DNA is heated so the two strands separate. (ii) Annealing — two sets of primers (short synthetic oligonucleotides complementary to the flanking regions) bind to the single strands. (iii) Extension — the enzyme DNA polymerase extends the primers using the supplied nucleotides and the template, building new strands. Repeating the cycle many times can amplify the segment about a billion times.
Q20. What is a bioreactor, and what is the purpose of the stirrer and the sparger in a stirred-tank type?
Answer: A bioreactor is a vessel in which raw materials are biologically converted into products using microbial, plant, animal or human cells, under optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). In a stirred-tank bioreactor the stirrer (agitator) ensures even mixing and distributes oxygen throughout the vessel; in the sparged type, sterile air is bubbled through to improve oxygen availability. The vessel also has temperature and pH control, a foam control system and sampling ports.
Analytical and Multi-Concept (Q21–Q30)
Q21. A piece of alien DNA is transferred into a bacterium but fails to multiply in the progeny cells. Explain the reason and the remedy.
Answer: An alien piece of DNA on its own has no way to replicate, because it lacks an origin of replication and is not part of a chromosome that can copy itself. The remedy is to link it to a vector (a plasmid or bacteriophage DNA) that carries an ori. Once joined to the ori, the alien DNA becomes part of a replicating unit, multiplies with the host, and is inherited by the progeny. This linking and multiplication is what we call cloning.
Q22. EcoRI cuts the palindrome GAATTC between G and A on both strands. Explain why fragments cut by EcoRI from two completely different genomes can still be joined together.
Answer: Because EcoRI recognises the same palindromic sequence wherever it occurs, every fragment it releases carries the identical single-stranded sticky end (an AATT overhang), regardless of which genome it came from. These complementary overhangs form hydrogen bonds with one another, so a fragment from one source can base-pair with a fragment from another. DNA ligase then seals the backbone, producing a recombinant molecule made of DNA from different sources.
Q23. On an agarose gel, one band lies close to the wells and another lies far from them. Which fragment is larger, at which end was the sample loaded, and why?
Answer: The band far from the wells is the smaller fragment, because the agarose sieve lets small fragments move faster and farther. The band close to the wells is the larger fragment. The sample was loaded at the cathode end (the wells), and the fragments migrated towards the anode, since DNA is negatively charged. Fragment size therefore maps directly onto distance travelled.
Q24. Why did scientists move from the two-antibiotic selection method to the blue-white (beta-galactosidase) method? Compare the two on the point of convenience.
Answer: In the two-antibiotic method, recombinants are found by plating on one antibiotic and then replica-plating on a second, because the insert inactivates one resistance gene. This needs simultaneous plating on two different plates and is laborious. The blue-white method inserts the foreign DNA into the beta-galactosidase gene, so recombinants show up as colourless colonies on a single plate with a chromogenic substrate while non-recombinants turn blue. Reading one plate instead of comparing two makes it far more convenient, which is why it is preferred.
Q25. DNA is a hydrophilic molecule. Explain how this single fact shapes the way we get recombinant DNA into a bacterial cell.
Answer: Because DNA is hydrophilic, it cannot cross the hydrophobic cell membrane freely, so a cell will not simply absorb a plasmid from its surroundings. This is exactly why the cell must be made competent — treatment with a divalent cation such as calcium increases the DNA's passage through wall pores, and a brief heat shock at 42 degrees C followed by ice drives uptake. The hydrophilic nature of DNA is therefore the underlying reason competence has to be engineered artificially rather than occurring on its own.
Q26. In PCR, why is an ordinary DNA polymerase unsuitable, and how does the choice of enzyme solve the problem?
Answer: Each PCR cycle begins with denaturation at a high temperature to separate the DNA strands. An ordinary DNA polymerase would be denatured and destroyed at that temperature, so fresh enzyme would be needed every cycle. The solution is a thermostable DNA polymerase, Taq polymerase, isolated from the bacterium Thermus aquaticus. It stays active through the high-temperature denaturation step, so the same enzyme can drive repeated cycles and amplify the DNA about a billion-fold.
Q27. After a recombinant protein has been synthesised inside cultured cells, several steps still remain before it can be sold. Describe these steps collectively and their purpose.
Answer: The steps after the biosynthetic stage are together called downstream processing. They include separation and purification of the product from the culture, formulation with suitable preservatives, and, for a drug, clinical trials. Each product must also pass strict quality-control testing. The purpose is to convert the crude biological output into a safe, stable, marketable finished product. Downstream processing varies from product to product.
Q28. Why can a small shake-flask culture not meet industrial demand, and how do a continuous culture system and a bioreactor overcome this?
Answer: Small-volume cultures cannot yield appreciable quantities of a product. In a continuous culture system, used medium is drained from one side while fresh medium is added from the other, keeping the cells in their physiologically most active log (exponential) phase; this produces a larger biomass and higher protein yield. A bioreactor scales this up further, processing 100 to 1000 litres of culture while supplying optimum temperature, pH, oxygen and nutrients. Together they turn a laboratory-scale process into an industrial one.
Q29. Arrange the following steps of recombinant DNA technology in the correct sequence and briefly justify the order: ligation into a vector; isolation of DNA; extraction of the product; culturing at large scale; cutting with restriction enzymes; transfer into the host.
Answer: The correct sequence is: (1) isolation of DNA, so the genetic material is pure and free of other molecules; (2) cutting with restriction enzymes, to release the gene of interest and open the vector at the same site; (3) ligation into a vector, to build the recombinant molecule; (4) transfer into the host, so the recombinant DNA can replicate; (5) culturing at large scale in a bioreactor to grow biomass and express the protein; and (6) extraction of the product through downstream processing. Each step supplies exactly what the next one needs.
Q30. A biotechnologist wants a human protein manufactured by bacteria. Trace the journey of the human gene from an intact cell all the way to a purified protein, naming the key tool used at each stage.
Answer: The cell is first broken open with lysozyme and the DNA purified (RNA removed by ribonuclease, protein by protease, DNA precipitated with chilled ethanol). The gene of interest is cut out with a restriction endonuclease, which also opens a vector such as pBR322 at the same site so their sticky ends match. DNA ligase joins gene and vector into recombinant DNA, and PCR with Taq polymerase can amplify the gene beforehand if needed. The recombinant DNA is introduced into competent E. coli (CaCl2 and a 42 degree C heat shock), and selectable markers identify the transformed, recombinant colonies. These are grown in a bioreactor under optimum conditions so the gene is expressed, and finally the protein is separated, purified and formulated by downstream processing.
End of Section 14
You have now worked through 30 examples spanning the principles of biotechnology, restriction enzymes and palindromes, gel electrophoresis, cloning vectors and pBR322 selection, plant and animal vectors, competent cells and DNA delivery, DNA isolation, the PCR reaction, bioreactors and downstream processing. Whenever a question touches selection or scale-up, remember the two habits — name both markers when you select a recombinant, and always link a tool to the exact job it does — and you will rarely leave marks on the table.