BiologyISC Class 12 · Biotechnology - Principles and Processes

Processes of Recombinant DNA Technology: ISC Class 12 Biology

Updated 15 October 2026 · Fact-checked

Recombinant DNA technology follows a sequence: isolate pure DNA, cut it and the vector with the same restriction enzyme, separate the gene by gel electrophoresis, amplify it by PCR, ligate it into the vector, transfer the recombinant DNA into a competent host, select and grow transformants in a bioreactor, and purify the product by downstream processing.

Understand Processes of Recombinant DNA Technology

The sequence (learn as a flow chart). Isolation of DNA → fragmentation by restriction endonucleases → isolation of the desired fragment → (amplification by PCR) → ligation into a vector → transfer of rDNA into the host → selection and culture of host cells on a large scale → extraction and purification of the product.

1. Isolation of genetic material (DNA). DNA must be pure, free of other macromolecules. Break cells open (lysis) with enzymes:

Source	Enzyme to break the cell
bacteria	lysozyme
plant cells	cellulase
fungi	chitinase

Then remove RNA with ribonuclease and proteins (such as histones) with protease; other molecules by suitable treatments. Adding chilled ethanol precipitates purified DNA as fine threads, removed by spooling.

2. Cutting at specific locations. Incubate pure DNA with the restriction enzyme at its optimal conditions; check progress by agarose gel electrophoresis. Repeat with the vector DNA using the same enzyme. Mix the gene of interest with the opened vector and add DNA ligase → recombinant DNA.

3. Isolating the gene: gel electrophoresis. DNA is negatively charged and moves towards the anode; the agarose gel (from seaweed) sieves fragments so smaller ones move farther. Stain with ethidium bromide, view under UV light → orange bands. Cut out the band and extract the DNA: elution.

4. Amplification by PCR (polymerase chain reaction), in vitro. Ingredients: template DNA, two sets of primers (short chemically synthesised oligonucleotides complementary to the ends of the target region), deoxynucleotides, and a thermostable DNA polymerase, Taq polymerase from Thermus aquaticus, which stays active at the high denaturation temperature.

Step	Approx. temperature	Event
Denaturation	about 94 °C	double strands separate
Annealing	about 50-60 °C	primers bind to complementary sequences
Extension	about 72 °C	Taq polymerase extends primers using nucleotides

Each cycle doubles the target: after n cycles, up to 2ⁿ copies per template; about 30 cycles give roughly a billion copies. The amplified fragment can be ligated into a vector. (Amplification in vivo = cloning by growing the host.)

5. Insertion of rDNA into the host. DNA is hydrophilic and cannot pass through membranes, so the host is made competent.

Chemical and heat shock: treat bacteria with a divalent cation such as calcium (CaCl₂); incubate with rDNA on ice, give a heat shock at 42 °C, put back on ice.
Microinjection: rDNA injected directly into the nucleus of an animal cell.
Biolistics (gene gun): plant cells bombarded with gold or tungsten micro-particles coated with DNA.
Disarmed pathogens: Agrobacterium (plants), retroviruses (animals).

Spreading cells on ampicillin plates keeps only transformants (amp^R is the selectable marker).

6. Obtaining the foreign gene product. The aim is usually a protein. A protein made from a gene expressed in a heterologous host is a recombinant protein. Small cultures give little product, so large-scale continuous culture is used: used medium drained from one side and fresh medium added from the other, keeping cells in the log (exponential) phase, for larger biomass and higher yield.

7. Bioreactors. Vessels of 100-1000 litres in which raw materials are biologically converted into specific products by microbial, plant, animal or human cells, under optimal conditions (temperature, pH, substrate, salts, vitamins, oxygen). A sterile (aseptic) environment is essential so that contaminating microbes do not grow and spoil the product.

Stirred-tank bioreactor	Sparged stirred-tank bioreactor
cylindrical or curved base for mixing; a stirrer gives even mixing and oxygen throughout	sterile air bubbles are sparged through the medium, increasing the area for oxygen transfer and helping mixing

Common parts (label in a diagram): motor and agitator (stirrer), sterile air inlet / sparger, foam breaker (foam control), temperature control (jacket), pH control, sampling port, culture broth, exhaust.

8. Downstream processing. After biosynthesis the product is separated and purified, then formulated with suitable preservatives. Drugs undergo clinical trials; strict quality control is needed. The steps differ for each product.

Growth arithmetic. Cells after g generations from N₀ = N₀ × 2^g, with g = time ÷ doubling time.

Processes of Recombinant DNA Technology: key facts

PCR yield: $copies = m \times 2^{n}$; m templates, n cycles (ideal doubling).
Bacterial growth: $N = N_{0} \times 2^{g}, g = \frac{time}{doubling time}$; Exponential, not g × N₀.
Enzymes and reagents for DNA isolation: Bacteria lysozyme
Plant cells cellulase
Fungi chitinase
RNA removed by ribonuclease
Proteins removed by protease
DNA precipitated by chilled ethanol (spooling); Match the wall-breaking enzyme to the cell type.
PCR cycle: 1.Denaturation, about 94 °C: strands separate
2.Annealing, about 50-60 °C: primers bind
3.Extension, about 72 °C: Taq polymerase extends the primers; Taq polymerase from Thermus aquaticus.

Bacteria	lysozyme
Plant cells	cellulase
Fungi	chitinase
RNA removed by	ribonuclease
Proteins removed by	protease
DNA precipitated by	chilled ethanol (spooling)

How to answer Processes of Recombinant DNA Technology questions

ISC asks the sequence of steps, PCR steps and Taq polymerase, competent hosts and transfer methods, bioreactor types and sterility, gel staining, downstream processing, and exponential-growth or PCR sums.

1Write the steps in order with arrows; each missing step loses a mark in 'flow chart' items.
2Name enzymes with their purpose: lysozyme/cellulase/chitinase for lysis, ribonuclease, protease, restriction enzyme, ligase, Taq polymerase.
3For PCR, give each step with its temperature and event; name the source of Taq polymerase in binomial form.
4For growth or PCR sums, find the number of doublings first, then use 2 to that power; never multiply by the number of doublings.
5For bioreactors, distinguish stirring from sparging, and say why sterility matters.
6Use 'ethidium bromide' for the stain and 'UV light' for viewing; they are different things.

Processes of Recombinant DNA Technology in real life

The idea, met in an everyday situation. Answer the question in your own words, then compare with the model answer. The one-line hook is what to remember.

PCR for early diagnosis

Suppose a blood sample from a patient with a suspected viral infection contains only about 5 copies of a viral gene in the tested volume, too few to detect directly.

How many copies would 20 PCR cycles produce, and why does this help diagnosis?

Try it first, then show the solutionSolution

Each cycle doubles the target, so copies = 5 × 2²⁰.
2²⁰ = 1 048 576, so copies = 5 242 880 (about 5 million).
This amount is easily detected, so infection can be confirmed before symptoms or antibodies appear.
The primers bind only the viral sequence, so the amplified DNA is specific to that virus.

Answer: About 5.2 million copies; PCR detects very low levels of a pathogen early and specifically.

Memory hook: Double, double: PCR finds the few.

Tricks to remember Processes of Recombinant DNA Technology

Short memory aids. Say each one aloud once, then use it on a question the same day.

I-C-I-A-L-T-S-E
Isolate, Cut, Isolate fragment, Amplify, Ligate, Transfer, Select and grow, Extract.
Use it when: Order of rDNA steps.
Hot, cool, warm
PCR goes hot (94 °C, separate), cool (50-60 °C, primers stick), warm (72 °C, Taq builds).
Use it when: PCR steps.
Ice, heat, ice
Calcium-treated cells: ice → 42 °C → ice.
Use it when: Heat-shock transformation.

Processes of Recombinant DNA Technology: solved examples

Ordered from board level to HOTS. Try each one before you open the solution.

Example 1: Isolating DNA · 3 marks · Short answerBoard level

How is pure DNA isolated from a plant cell? Name the enzymes used and the reagent that precipitates DNA.

Try it first, then show the solutionSolution

Cell walls are broken with cellulase to release DNA along with RNA, proteins, polysaccharides and lipids (1 mark).
RNA is removed with ribonuclease and proteins (histones) with protease; other molecules by suitable treatments (1 mark).
Chilled ethanol is added; purified DNA precipitates as fine threads, collected by spooling (1 mark).

Answer: Cellulase, ribonuclease, protease; chilled ethanol precipitates DNA.

Example 2: PCR steps · 3 marks · Short answerBoard level

Describe the three steps of one PCR cycle and explain why Taq polymerase is used.

Try it first, then show the solutionSolution

Denaturation at about 94 °C separates the two DNA strands (1 mark).
Annealing at about 50-60 °C lets the two primers bind to complementary sequences; extension at about 72 °C lets the polymerase extend the primers using deoxynucleotides (1 mark).
Taq polymerase from Thermus aquaticus is thermostable and stays active through repeated high-temperature denaturation, so it need not be added every cycle (1 mark).

Answer: Denaturation, annealing, extension; Taq is heat-stable.

Example 3: Protein from a growing culture · 2 marks · Short answerHarder board level

A transformed bacterium makes 2 molecules of a recombinant protein per cell. It divides every 30 minutes. Starting from one cell, how many protein molecules are present after 4 hours (assume none is lost)?

Try it first, then show the solutionSolution

4 hours = 240 min ÷ 30 = 8 generations; cells = 2⁸ = 256 (1 mark).
Protein = 256 × 2 = 512 molecules (1 mark).

Answer: 512 molecules.

Example 4: Sterility in a bioreactor · 3 marks · Short answerHarder board level

Assertion: A bioreactor must be kept sterile. Reason: Contaminating microbes would compete with the production cells and could spoil the product. Evaluate both and state the relationship. Name two systems in a stirred-tank bioreactor that maintain optimal conditions.

Try it first, then show the solutionSolution

Both are true (1 mark).
The reason explains the assertion: sterile (aseptic) conditions keep out unwanted microbes, which would use nutrients and contaminate the product (1 mark).
Systems: temperature control, pH control, oxygen delivery (sparger), foam control, agitator, sampling ports (any two) (1 mark).

Answer: Both true; R explains A; e.g. temperature and pH control systems.

Example 5: Making a host competent · 3 marks · Short answerHarder board level

Why must E. coli be made competent before transformation? Describe the heat-shock method and name one method each for plant and animal cells.

Try it first, then show the solutionSolution

DNA is hydrophilic and cannot pass through the cell membrane on its own (½ mark).
Cells are treated with a divalent cation such as calcium, incubated with rDNA on ice, given a brief heat shock at 42 °C and returned to ice; the DNA enters (1½ marks).
Plants: biolistics (gene gun) with gold or tungsten particles; animals: microinjection into the nucleus (1 mark).

Answer: Hydrophilic DNA needs help; Ca²⁺ + ice-42 °C-ice; gene gun; microinjection.

Example 6: Planning production · 4 marks · Short answerHOTS / competency

Suppose a company must produce a recombinant enzyme from E. coli. (a) Why is a continuous culture better than a batch culture? (b) Why would a sparged stirred-tank reactor suit a fast-growing aerobic culture? (c) List three steps of downstream processing before the enzyme is sold.

Try it first, then show the solutionSolution

(a) Draining used medium and adding fresh medium keeps cells in the log (exponential) phase, giving larger biomass and higher yield (1 mark).
(b) Sterile air bubbles increase the area for oxygen transfer and help mixing, meeting the high oxygen demand of dense aerobic cells (1 mark).
(c) Separation of the product from cells and medium; purification; formulation with preservatives, followed by quality control testing (2 marks).

Answer: (a) Keeps log phase, higher yield. (b) Better oxygen transfer. (c) Separate, purify, formulate, quality test.

Where students lose marks in Processes of Recombinant DNA Technology

The slips examiners see most often, and what to write instead.

Mistake: Linear instead of exponential growth
In 2025 examiners reported that candidates found 9 doublings but answered 9 molecules instead of 2⁹ = 512.
Do this: Number of cells = 2^(number of generations).
Usually costs: 1 mark.
Mistake: Writing UV rays for the stain
In 2024 examiners saw 'UV rays' instead of the chemical, and 'ethylene bromide' for ethidium bromide.
Do this: Stain: ethidium bromide (chemical); view: UV light (radiation).
Usually costs: 1 mark.
Mistake: Wrong logic on bioreactor sterility
Examiners (2024) noted confusion between options (b) and (d) on the statement that sterile conditions are not needed, and suggested clarifying 'sterile' and 'aseptic'.
Do this: A bioreactor must be sterile; sterility prevents, not promotes, unwanted microbes.
Usually costs: 1 mark.
Mistake: Misspelling Thermus aquaticus
In 2025 examiners reported made-up names such as 'Thermophilic aquaticus' or 'Taqupolitica', or methanogens.
Do this: Taq = Thermus aquaticus.
Usually costs: 1 mark.

Exam tips

Use arrows and number the steps in flow-chart answers.
Give the three PCR temperatures as approximate values.
Say 'divalent cation such as calcium' and '42 °C' for heat shock.
Learn the bioreactor parts list for a labelling question.
Show the 2^n step in every growth or PCR sum.

Practice questions on Processes of Recombinant DNA Technology

Write each answer on paper first. Open the hint only if you are stuck, and the answer only when you have finished.

Q1 · 1 mark · MCQBoard level
The chemical used to stain DNA in agarose gels is:
(a) ethylene bromide (b) ethidium bromide (c) X-gal (d) chilled ethanol
Hint
Orange bands under UV.
Answer
(b) ethidium bromide.
Q2 · 2 marks · Short answerBoard level
What is elution? Why are DNA bands visible only after staining?
Hint
Cutting out the band.
Answer
Elution is cutting out the separated DNA band from the agarose gel and extracting the DNA from the gel piece. Pure DNA cannot be seen in visible light; it must be stained with ethidium bromide and exposed to UV light to show orange bands.
Q3 · 1 mark · Assertion-reasonHarder board level
Assertion (A): In PCR, the DNA polymerase need not be replaced after every cycle.
Reason (R): Taq polymerase, from Thermus aquaticus, remains active at the high temperature of denaturation.
(a) Both A and R are true and R is the correct explanation of A.
(b) Both A and R are true but R is not the correct explanation of A.
(c) A is true but R is false.
(d) A is false but R is true.
Hint
Thermostable.
Answer
(a).
Q4 · 2 marks · Short answerHarder board level
A single template molecule is placed in a PCR machine. Calculate the copies present after 10 cycles, and the least number of cycles that yields one million copies or more.
Hint
2¹⁰; 2²⁰.
Answer
2¹⁰ = 1024 copies. 2¹⁹ = 524 288 is too few and 2²⁰ = 1 048 576 is enough, so 20 cycles.
Q5 · 2 marks · Short answerHarder board level
A flask is inoculated with 1000 transformed bacteria whose doubling time is 20 minutes. Calculate the population at the end of a 2-hour run.
Hint
6 generations.
Answer
120 ÷ 20 = 6 generations; 1000 × 2⁶ = 1000 × 64 = 64 000 cells.
Q6 · 2 marks · Short answerHarder board level
Distinguish between a simple stirred-tank and a sparged stirred-tank bioreactor.
Hint
Bubbles.
Answer
In a simple stirred-tank reactor a stirrer mixes the contents and distributes oxygen. In a sparged stirred-tank reactor sterile air bubbles are also passed (sparged) through the medium, increasing the surface area for oxygen transfer.
Q7 · 3 marks · Short answerHOTS / competency
Suppose a student isolates DNA from a fungus using lysozyme, protease and chilled ethanol, and gets almost no DNA threads. Identify the error and explain.
Hint
Fungal walls contain chitin.
Answer
Lysozyme breaks bacterial cell walls, not fungal walls, which contain chitin. The cells did not lyse, so little DNA was released for protease treatment and precipitation. She should use chitinase to break the fungal cell walls (then ribonuclease and protease, and chilled ethanol).
Q8 · 3 marks · Case-basedHOTS / competency
Read and answer.
Suppose a transformed bacterium carries a gene that makes one molecule of protein Y per cell. It divides every 15 minutes and is grown for 2 hours.
(a) How many generations and cells are there? (1)
(b) How many molecules of Y are made? (1)
(c) A student answers '8 molecules'. What mistake has she made? (1)
Hint
Exponential growth.
Answer
(a) 120 ÷ 15 = 8 generations; 2⁸ = 256 cells. (b) 256 molecules. (c) She counted the number of doublings (8) instead of applying exponential growth (2⁸).

Test your recall

Answer in your head or on paper first, then open the card. Pulling an answer out of memory makes it stick far better than re-reading.

1.Which enzyme breaks bacterial cell walls for DNA isolation?Show answer
Lysozyme.
2.What precipitates purified DNA?Show answer
Chilled ethanol.
3.Source of Taq polymerase?Show answer
Thermus aquaticus.
4.Temperature of heat shock in transformation?Show answer
42 °C.
5.Volume range of bioreactors?Show answer
100-1000 litres.
6.What is a recombinant protein?Show answer
A protein produced from a gene expressed in a heterologous host.

Processes of Recombinant DNA Technology: frequently asked questions

What are the main steps of recombinant DNA technology?

Isolation of DNA, cutting with restriction enzymes, isolating the gene, amplification by PCR, ligation into a vector, transfer into a competent host, selection and large-scale culture, and downstream processing.

What are the steps of PCR?

Denaturation (about 94 °C), annealing of primers (about 50-60 °C) and extension by Taq polymerase (about 72 °C), repeated many times.

Why are bioreactors used?

To grow large volumes of cells under optimal, sterile conditions so that products are made in large quantities.

What is downstream processing?

The separation, purification and formulation of the product after biosynthesis, followed by quality control (and clinical trials for drugs).

How is DNA introduced into plant cells?

By biolistics (gene gun) with DNA-coated gold or tungsten particles, or by a disarmed Agrobacterium Ti plasmid.