Biotechnology: Principles and Processes Class 12: Concepts, Diagrams and Board Answers

What Biotechnology: Principles and Processes is about

Weightage: This chapter is part of Unit IX, Biotechnology and its Applications, which carries 12 of the 70 theory marks together with Biotechnology and its Applications.Source: CBSE Biology (044) syllabus 2026-27, course structure, Unit IX Biotechnology and its Applications

In real board papers: asked in 8 of the 8 board papers we hold (2023 to 2026), on average 7.8 marks per paper. See the questions

Key concepts

Principles of biotechnology and the first recombinant DNA

NCERT section 9.1 Principles of Biotechnology

Why genetic engineering? Sexual reproduction gives variation, asexual reproduction preserves information. Traditional hybridisation in plant and animal breeding often carries undesirable genes along with the desired ones. Genetic engineering (recombinant DNA, gene cloning, gene transfer) lets us introduce only one or a set of desirable genes.

Fate of alien DNA: a piece of DNA put into a new organism usually cannot multiply in the progeny cells. It can multiply only if it becomes part of a chromosome or is linked to a DNA sequence called the origin of replication, which starts replication. Linking alien DNA to an origin so that it makes many identical copies is cloning.

First recombinant DNA (Stanley Cohen and Herbert Boyer, 1972):
1. An antibiotic resistance gene was cut out of a plasmid of Salmonella typhimurium using restriction enzymes ('molecular scissors').
2. It was linked to a plasmid (an autonomously replicating, circular, extra-chromosomal DNA) acting as a vector, by DNA ligase.
3. This new circular, self-replicating DNA made in vitro is recombinant DNA.
4. It was transferred into E. coli (closely related to Salmonella), where it replicated using the host's DNA polymerase: cloning of the antibiotic resistance gene in E. coli.

(Boyer had found restriction enzymes that leave 'sticky ends'; Cohen had a method of removing plasmids and putting them back into cells.)

Three basic steps of genetically modifying an organism:
1. Identify DNA with desirable genes.
2. Introduce the identified DNA into the host.
3. Maintain the introduced DNA in the host and transfer it to the progeny.

Worked example

Why can a piece of foreign DNA introduced into a bacterium not multiply on its own? How did Cohen and Boyer overcome this? (2 marks)

Try it first, then show the solutionSolution

1. Replication starts only at a specific sequence, the origin of replication; a bare piece of DNA without one cannot be copied in progeny cells. (1)
2. They joined the gene to a plasmid vector (which has an origin of replication) using restriction enzymes and DNA ligase, then put it into E. coli, where it replicated with the plasmid. (1)

Answer: It lacks an origin of replication; linking it to a plasmid vector (with ori) lets it replicate.

Restriction enzymes and sticky ends

NCERT section 9.2.1 Restriction Enzymes

Discovery: in 1963, two enzymes that restrict the growth of bacteriophage in E. coli were isolated: one added methyl groups to DNA, the other cut DNA (the restriction endonuclease). The first sequence-specific one, Hind II, was isolated about five years later; it always cuts at a particular point after recognising a six-base-pair recognition sequence. Today more than 900 restriction enzymes from over 230 strains of bacteria are known.

Naming (EcoRI):

• E = genus Escherichia; co = species coli; R = strain RY 13; I (Roman numeral) = the order in which the enzyme was isolated from that strain.

Nucleases:

• Exonucleases remove nucleotides from the ends of DNA.
• Endonucleases cut at specific positions within DNA. Restriction enzymes are endonucleases.

How they cut: each enzyme 'inspects' DNA, finds its recognition sequence, binds and cuts both strands at specific points in the sugar-phosphate backbone.

Palindromes: the recognition sequence is a palindrome: the base sequence reads the same on both strands when read in the same direction (5′→3′ on each). Example (EcoRI site):
5′-GAATTC-3′
3′-CTTAAG-5′

Sticky ends: the enzyme cuts a little away from the centre of the palindrome, but between the same two bases on both strands (EcoRI cuts between G and A). This leaves single-stranded overhangs called sticky ends, which form hydrogen bonds with complementary cut ends. This stickiness helps DNA ligase join them.

Key rule: the vector and the source DNA must be cut with the same restriction enzyme, so both have matching sticky ends that can be joined by ligase into recombinant DNA.

Worked example

Write the strand complementary to 5′-GAATTC-3′ and show why it is a palindrome. EcoRI cuts between G and A on both strands; draw the sticky ends produced. (3 marks)

Try it first, then show the solutionSolution

1. Complementary strand (aligned): 3′-CTTAAG-5′. Read 5′→3′ it is GAATTC, the same as the top strand: a palindrome. (1)
2. Cutting between G and A on both strands gives: left fragment 5′-G-3′ / 3′-CTTAA-5′ and right fragment 5′-AATTC-3′ / 3′-G-5′. (1)
3. Each fragment has a single-stranded AATT overhang (sticky end) that can pair with the complementary overhang of any DNA cut by EcoRI. (1)

Answer: Both strands read GAATTC (5′→3′); EcoRI leaves 5′-AATT overhangs (sticky ends).

Gel electrophoresis and isolation of DNA fragments

NCERT section 9.2.1 (Separation and isolation of DNA fragments)

Purpose: separate the DNA fragments produced by restriction digestion, and check whether digestion has happened.

Principle: DNA is negatively charged, so in an electric field it moves towards the anode (positive electrode) through a matrix. The most common matrix is agarose, a natural polymer from seaweeds. The gel acts as a sieve: smaller fragments move farther; larger ones stay nearer the wells (where the sample was loaded).

Steps:
1. Load the DNA samples into wells at the cathode end of an agarose gel.
2. Pass current; fragments separate by size.
3. Stain with ethidium bromide and expose to UV light: DNA appears as bright orange bands (pure DNA cannot be seen in visible light without staining).
4. Cut out the desired band and extract the DNA from the gel piece: elution.
5. The purified fragments are used to build recombinant DNA by joining with vectors.

Reading a gel: an undigested sample shows one large band near the well; digested samples show several bands; the band farthest from the well is the smallest fragment.

Worked example

A DNA sample is digested and run on an agarose gel. Bands are seen at 2 cm, 4 cm and 7 cm from the wells. Which band has the smallest fragment and why? How are the bands seen, and how is a band recovered? (3 marks)

Try it first, then show the solutionSolution

1. The band at 7 cm is the smallest, because the agarose sieve lets smaller fragments move farther towards the anode. (1)
2. Bands are seen after staining with ethidium bromide and exposing the gel to UV light (orange bands). (1)
3. The desired band is cut out of the gel and the DNA extracted from it (elution). (1)

Answer: 7 cm band (smallest); ethidium bromide + UV; elution.

Cloning vectors and pBR322

NCERT section 9.2.2 Cloning Vectors

Plasmids and bacteriophages replicate in bacteria independently of chromosomal DNA. Phages have very high copy numbers; plasmids have 1–2 or 15–100 (even more) copies per cell. Linking alien DNA to them multiplies it to their copy number. Modern vectors are engineered for easy linking and easy selection.

Features of a cloning vector:
1. Origin of replication (ori): where replication starts; also controls the copy number of linked DNA. For many copies, use a vector whose ori supports a high copy number.
2. Selectable marker: identifies and eliminates non-transformants and lets transformants grow. Genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are useful for E. coli (normal E. coli lacks these resistances). (Transformation: introducing a piece of DNA into a host bacterium.)
3. Cloning sites: very few, preferably single, recognition sites for common restriction enzymes; several sites would cut the vector into many fragments and complicate cloning.
4. Vectors for plants and animals (see the next concept).

pBR322 (an E. coli vector): has ori, rop (codes for proteins involved in plasmid replication), amp^R and tet^R genes, and restriction sites EcoR I, Hind III, Cla I, BamH I, Sal I, Pst I, Pvu II.

Selection by insertional inactivation of antibiotic resistance:
1. Insert foreign DNA at the BamH I site in tet^R: recombinant plasmids lose tetracycline resistance but keep ampicillin resistance.
2. Plate transformants on ampicillin: only cells with a plasmid (recombinant or not) grow.
3. Replica-plate them on tetracycline: recombinants do not grow; non-recombinants grow on both.
This needs two plates at once, so it is cumbersome.

Blue-white selection (insertional inactivation of β-galactosidase):

• Foreign DNA is inserted into the coding sequence of β-galactosidase, inactivating it (insertional inactivation).
• With a chromogenic substrate: plasmids without an insert make β-galactosidase → blue colonies; recombinants (insert present) cannot → colourless (white) colonies.

Worked example

A gene is inserted at the BamH I site of pBR322 and the plasmids are put into E. coli. Predict the growth on ampicillin and on tetracycline plates of (a) untransformed cells, (b) cells with non-recombinant pBR322, (c) cells with recombinant pBR322. (3 marks)

Try it first, then show the solutionSolution

1. (a) Untransformed cells have no resistance: no growth on either plate. (1)
2. (b) Non-recombinant plasmids have intact amp^R and tet^R: grow on both plates. (1)
3. (c) The insert at BamH I inactivates tet^R, but amp^R works: grow on ampicillin, not on tetracycline. (1)

Answer: (a) neither (b) both (c) ampicillin only; recombinants are those that grow on ampicillin but not tetracycline.

Vectors for plants and animals and competent hosts

NCERT section 9.2.2 (iv) Vectors for cloning genes in plants and animals; 9.2.3 Competent Host

Learning from pathogens: bacteria and viruses have long known how to deliver genes into eukaryotic cells.

• Agrobacterium tumefaciens, a pathogen of several dicot plants, delivers a piece of DNA called T-DNA that turns normal plant cells into a tumour making chemicals the pathogen needs. Its Ti (tumour-inducing) plasmid has been modified into a cloning vector that is no longer pathogenic but still delivers genes of interest into many plants.
• Retroviruses can turn normal animal cells cancerous; disarmed retroviruses now deliver desirable genes into animal cells.

Competent host (making bacteria take up DNA): DNA is hydrophilic and cannot pass through cell membranes.
1. Treat bacterial cells with a specific concentration of a divalent cation, such as calcium, which increases DNA entry through pores in the cell wall.
2. Incubate cells with recombinant DNA on ice.
3. Heat shock: place them briefly at 42 °C.
4. Put them back on ice. The bacteria take up the recombinant DNA.

Other methods of gene transfer:

• Microinjection: recombinant DNA injected directly into the nucleus of an animal cell.
• Biolistics (gene gun): plant cells bombarded with high-velocity micro-particles of gold or tungsten coated with DNA.
• Disarmed pathogen vectors: allowed to infect the cell and transfer the recombinant DNA.

Worked example

Suggest the most suitable method to introduce a gene into (a) a plant cell, (b) an animal egg nucleus, (c) E. coli. Explain the method for (c). (3 marks)

Try it first, then show the solutionSolution

1. (a) Biolistics (gene gun) with DNA-coated gold or tungsten particles, or a disarmed Ti plasmid of Agrobacterium. (½)
2. (b) Microinjection of DNA directly into the nucleus. (½)
3. (c) Make E. coli competent by treating with calcium (a divalent cation), incubate with the recombinant DNA on ice, (1)
4. give a brief heat shock at 42 °C, and return to ice so the cells take up the DNA. (1)

Answer: (a) Gene gun or Ti plasmid (b) microinjection (c) Ca²⁺ treatment, ice, heat shock at 42 °C, ice.

Isolating DNA and cutting it

NCERT section 9.3 Processes of Recombinant DNA Technology (9.3.1 Isolation of DNA; 9.3.2 Cutting of DNA)

Overview of recombinant DNA technology: isolation of DNA → fragmentation by restriction endonucleases → isolation of the desired fragment → ligation into a vector → transfer into the host → culturing host cells on a large scale → extraction of the desired product.

Isolation of DNA (it must be pure for restriction enzymes to work):
1. Break open the cells to release DNA with RNA, proteins, polysaccharides and lipids, using enzymes:

• Lysozyme for bacteria.
• Cellulase for plant cells.
• Chitinase for fungi.

2. Genes lie on long DNA molecules wound with proteins such as histones. Remove RNA with ribonuclease and proteins with protease; other molecules by suitable treatments.
3. Add chilled ethanol: purified DNA precipitates as a collection of fine threads, which can be removed by spooling.

Cutting DNA at specific locations:
1. Incubate purified DNA with the restriction enzyme at the optimal conditions for that enzyme.
2. Check the progress of digestion by agarose gel electrophoresis (DNA moves to the anode).
3. Repeat with the vector DNA.
4. Mix the cut-out gene of interest with the cut vector and add ligase: recombinant DNA forms.

Counting fragments: cutting a linear DNA at n sites gives n + 1 fragments; cutting a circular DNA (plasmid) at n sites gives n fragments.

Worked example

Which enzymes would you use to release DNA from (a) a bacterium, (b) a plant cell, (c) a fungus? How are RNA and proteins removed, and how is the DNA finally obtained? (3 marks)

Try it first, then show the solutionSolution

1. (a) Lysozyme (b) cellulase (c) chitinase, which break the cell walls. (1)
2. RNA is removed with ribonuclease and proteins (including histones) with protease. (1)
3. Adding chilled ethanol precipitates purified DNA as fine threads, collected by spooling. (1)

Answer: Lysozyme, cellulase, chitinase; RNase and protease; precipitate with chilled ethanol and spool.

Polymerase chain reaction (PCR)

NCERT section 9.3.3 Amplification of Gene of Interest using PCR

PCR makes multiple copies of a gene (DNA) of interest in vitro using:

• two sets of primers: small, chemically synthesised oligonucleotides complementary to regions flanking the target DNA;
• the enzyme DNA polymerase, which extends the primers using nucleotides supplied and the genomic DNA as template.

Each cycle has three steps:
1. Denaturation: high temperature separates the two DNA strands.
2. Primer annealing: primers bind to their complementary regions on the single strands.
3. Extension: DNA polymerase extends the primers, making new strands.

Thermostable polymerase: repeated cycles need an enzyme that stays active through the high-temperature denaturation: Taq polymerase, from the bacterium Thermus aquaticus.

Amplification: the number of copies doubles each cycle, so one DNA molecule gives 2ⁿ copies after n cycles; repeated many times, the segment is amplified about a billion times (2³⁰ ≈ 1.07 × 10⁹).

Uses in this unit: amplified fragments can be ligated into vectors for cloning; in Chapter 5, PCR made DNA fingerprinting possible from a single cell; in Chapter 10, PCR detects pathogens and mutations early.

Worked example

Starting with 4 double-stranded copies of a target DNA, how many copies will there be after 10 PCR cycles? Why must the polymerase be thermostable? (2 marks)

Try it first, then show the solutionSolution

1. Copies double each cycle: 4 × 2¹⁰ = 4 × 1024 = 4096. Check by doubling: 4 → 8 → 16 → 32 → 64 → 128 → 256 → 512 → 1024 → 2048 → 4096 (10 doublings). (1)
2. Each cycle begins with high-temperature denaturation; Taq polymerase from Thermus aquaticus stays active at these temperatures, so it need not be added again every cycle. (1)

Answer: 4096 copies; the polymerase must survive the denaturation temperature (Taq polymerase).

Transformation, expression, bioreactors and downstream processing

NCERT section 9.3.4 Insertion of Recombinant DNA into the Host; 9.3.5 Obtaining the Foreign Gene Product; 9.3.6 Downstream Processing

Insertion and selection: competent recipient cells take up DNA from their surroundings. If recombinant DNA carries an ampicillin resistance gene, transformed E. coli become ampicillin-resistant. On agar plates with ampicillin, only transformants grow; untransformed cells die. The ampicillin resistance gene is a selectable marker.

Obtaining the foreign gene product: the usual aim is a protein. A protein-coding gene expressed in a heterologous (different) host gives a recombinant protein. Cells are first grown on a small scale; then continuous culture (used medium drained from one side, fresh medium added from the other) keeps cells in their most active log/exponential phase, giving more biomass and higher yields.

Bioreactors: small cultures cannot give appreciable amounts, so bioreactors process large volumes (100–1000 litres). They are vessels in which raw materials are biologically converted into specific products using microbial, plant, animal or human cells, under optimum conditions (temperature, pH, substrate, salts, vitamins, oxygen).

• Most common: stirred-tank bioreactors, usually cylindrical or with a curved base for good mixing. The stirrer gives even mixing and oxygen availability; alternatively, air is bubbled (sparged) through.
• Parts: agitator system, oxygen delivery system, foam control system, temperature control system, pH control system and sampling ports.
• Advantages over shake flasks: besides better aeration and mixing, they allow control of temperature, pH and foam, sampling, and much larger volumes.

Downstream processing: after biosynthesis, the product is separated and purified, formulated with preservatives, put through clinical trials (for drugs) and strict quality control. These steps vary from product to product.

Worked example

List the systems of a stirred-tank bioreactor and state what downstream processing includes. (3 marks)

Try it first, then show the solutionSolution

1. Agitator (stirrer) system, oxygen delivery system (or sparging of sterile air) and foam control system. (1)
2. Temperature control system, pH control system and sampling ports to withdraw small volumes. (1)
3. Downstream processing: separation and purification of the product, formulation with preservatives, clinical trials (for drugs) and strict quality control. (1)

Answer: Agitator, oxygen delivery, foam control, temperature and pH control, sampling ports; separation, purification, formulation, trials and quality control.

Biotechnology: Principles and Processes in real life

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

Topics in this chapter

• Restriction Enzymes
• Cloning Vectors and pBR322
• Processes of Recombinant DNA Technology

Biotechnology: Principles and Processes: key facts

Restriction enzyme naming

$\text{EcoRI}:E=\text{Escherichia};co=\text{coli};R=\text{strain RY 13};I=\text{first isolated}$

Genus (1 letter), species (2 letters), strain, Roman numeral.

EcoRI site and cut

$5^{\prime }\text{-G}\downarrow \text{AATTC-}{3}^{\prime };3^{\prime }\text{-CTTAA}\uparrow \text{G-}{5}^{\prime }$

Palindrome; leaves AATT sticky ends.

PCR amplification

$\text{copies after }n\text{ cycles}=N_0\times 2^n;2^{30}\approx 1.07\times 10^9$

Each cycle: denaturation, annealing, extension.

Fragments from cutting

$\text{linear DNA: }n\text{ cuts}\to n+1\text{ fragments};\text{circular DNA: }n\text{ cuts}\to n\text{ fragments}$

Useful for gel-band questions.

Cell-wall enzymes for DNA isolation

$\text{bacteria: lysozyme};\text{plants: cellulase};\text{fungi: chitinase}$

Then RNase, protease, chilled ethanol.

Selection in pBR322 (insert in tet^R)

${\text{recombinant: amp}}^{+}{\text{tet}}^{-};{\text{non-recombinant: amp}}^{+}{\text{tet}}^{+};{\text{untransformed: amp}}^{-}{\text{tet}}^{-}$

Blue-white: blue = no insert; colourless = recombinant.

Competent cells

${\text{Ca}}^{2+}\to \text{ice}\to 42{}^{\circ }\text{C heat shock}\to \text{ice}$

Divalent cation increases DNA entry.

Biotechnology: Principles and Processes: diagrams to practise

Practise each one on paper: draw it from the checklist, then tick off every label.

How the board asks Biotechnology: Principles and Processes

The question patterns to expect, and the method that works for each.

Tricks to remember Biotechnology: Principles and Processes

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

• Tools of rDNA: 'Real Lab Very Handy Pipettes'

  Restriction enzymes, Ligase, Vectors, Host, Polymerase.

  Use it when: Listing the tools of genetic engineering.

• EcoRI = 'E co R I': Genus-species-strain-number

  Escherichia coli RY13, first enzyme isolated.

  Use it when: Naming questions.

• Exo = Ends, Endo = Inside

  Exonucleases nibble from the ends; endonucleases cut within.

  Use it when: Types of nucleases.

• PCR: 'Dance, Attach, Extend' (D-A-E)

  Denaturation (separate strands), Annealing (primers stick), Extension (Taq adds nucleotides).

  Use it when: PCR steps.

• Wall breakers: 'Lazy Bacteria, Cellular Plants, Chitty Fungi'

  Lysozyme–bacteria; cellulase–plant cells; chitinase–fungi.

  Use it when: DNA isolation.

• Gel: 'Run to Red' (DNA runs to the positive anode)

  Negative DNA moves to the anode; small fragments run farthest.

  Use it when: Electrophoresis questions.

Biotechnology: Principles and Processes: board-level solved questions

Ordered from board level to HOTS. Cover the solution, write your own, then compare step by step.

Case-study practice (Section D style)

Read the situation, then answer every part. In the board paper this is a 4-mark question split into 1 + 2 + 1 marks.

Where the marks are

How a typical answer is split into step marks. Show every step: a right final answer alone rarely earns full marks.

Where students lose marks in Biotechnology: Principles and Processes

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

• ✗Mistake: Writing a palindrome by reading one strand backwards

  Wrong working

  5′-GAATTC-3′ is a palindrome because GAATTC reversed is CTTAAG.

  Right working

  It is a palindrome because the complementary strand, read 5′→3′, is also GAATTC.

  ✓Do this: Compare the two strands, each read 5′→3′.

  Usually costs: 1 mark.

• ✗Mistake: Saying DNA moves to the cathode in electrophoresis

  ✓Do this: DNA is negatively charged and moves towards the anode (positive electrode).

  Usually costs: ½ to 1 mark.

• ✗Mistake: Mixing up which colonies are recombinant in blue-white screening

  ✓Do this: Insert inactivates β-galactosidase → no colour → recombinant; blue colonies have no insert.

  Usually costs: 1 mark.

• ✗Mistake: Saying recombinants grow on tetracycline when the insert is at BamH I

  ✓Do this: BamH I lies in tet^R, so the insert destroys tetracycline resistance; recombinants grow on ampicillin only.

  Usually costs: 1 mark.

• ✗Mistake: Forgetting why Taq polymerase is used

  ✓Do this: It is thermostable (from Thermus aquaticus) and stays active during high-temperature denaturation in every cycle.

  Usually costs: 1 mark.

• ✗Mistake: Cutting the vector and the gene with different enzymes

  ✓Do this: Both must be cut with the same restriction enzyme so their sticky ends match and ligase can join them.

  Usually costs: 1 mark.

NCERT exercise guide: Biotechnology: Principles and Processes

What each exercise in the NCERT chapter tests, how to approach it, and a question of the same kind solved step by step. Questions here are written in our own words.

Biotechnology: Principles and Processes: previous year questions

• Asked in 8 of the 8 board papers we hold (2023 to 2026), on average 7.8 marks per paper; 34 questions in all, counting OR alternatives. In 4 of 4 sample papers.

Counted from the real papers on our previous year papers page, not estimated. Average marks are over every board paper we hold, with an OR pair counted once. Chapter tags are ours.

Solved board questions from Biotechnology: Principles and Processes

Real questions from recent papers. Try each one first, then open our step-by-step solution with marks per step.

Every Biotechnology: Principles and Processes question, by year

Practice questions on Biotechnology: Principles and Processes

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

1. Q1 · 1 mark · MCQBoard level

   In gel electrophoresis, DNA fragments move towards the:
   (a) cathode, because DNA is positive (b) anode, because DNA is negative (c) cathode, because DNA is negative (d) anode, because DNA is positive

   Hint

   Phosphate groups.

   Answer

   (b) anode, because DNA is negative

2. Q2 · 1 mark · Assertion-reasonBoard level

   Assertion (A): Recombinant colonies appear colourless on a medium with a chromogenic substrate.
   Reason (R): The insert inactivates the gene for β-galactosidase.
   (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

   Insertional inactivation.

   Answer

   (a)

3. Q3 · 1 mark · MCQHarder board level

   Chitinase would be used to isolate DNA from:
   (a) bacteria (b) plant cells (c) fungi (d) animal cells

   Hint

   Cell wall made of chitin.

   Answer

   (c) fungi

4. Q4 · 2 marks · Very short answerBoard level

   State two differences between exonucleases and endonucleases. (2 marks)

   Hint

   Ends versus inside.

   Answer

   Exonucleases remove nucleotides from the ends of DNA; endonucleases cut at specific positions within DNA (1). Restriction enzymes are endonucleases that recognise specific (palindromic) sequences; exonucleases do not need an internal recognition site (1).

5. Q5 · 2 marks · Very short answerHarder board level

   What are the roles of ori and rop in pBR322? Why should a vector have only one site for a given restriction enzyme? (2 marks)

   Hint

   Copy number; fragments.

   Answer

   ori is where replication starts and controls copy number; rop codes for proteins involved in plasmid replication (1). Several sites would cut the vector into many fragments, complicating cloning (1).

6. Q6 · 2 marks · Very short answerBoard level

   Describe how DNA fragments are made visible after electrophoresis and how a desired fragment is recovered. (2 marks)

   Hint

   Stain + UV; elution.

   Answer

   The gel is stained with ethidium bromide and exposed to UV light; DNA shows as bright orange bands (1). The desired band is cut out and the DNA extracted from the gel piece (elution) for use in recombinant DNA (1).

7. Q7 · 3 marks · Short answerHarder board level

   Explain three methods of introducing recombinant DNA into host cells other than chemical transformation. (3 marks)

   Hint

   Injection, gun, pathogen.

   Answer

   Microinjection: recombinant DNA injected directly into the nucleus of an animal cell (1). Biolistics (gene gun): plant cells bombarded with high-velocity gold or tungsten micro-particles coated with DNA (1). Disarmed pathogen vectors (Ti plasmid of Agrobacterium, disarmed retroviruses) infect the cell and transfer the recombinant DNA (1).

8. Q8 · 3 marks · Short answerHarder board level

   What is meant by a selectable marker? Explain with the example of ampicillin resistance how transformants are selected. (3 marks)

   Hint

   Only transformants survive.

   Answer

   A selectable marker is a gene that helps identify and eliminate non-transformants and lets transformants grow (1). If recombinant DNA carries the ampicillin resistance gene, transformed E. coli become ampicillin-resistant (1). On agar with ampicillin, only transformants grow; untransformed cells die (1).

9. Q9 · 5 marks · Long answerHarder board level

   (a) Draw and label a sparged stirred-tank bioreactor. (3)
   (b) Explain what downstream processing involves and why quality control is essential. (2) (5 marks)

   Hint

   Six systems.

   Answer

   (a) Cylindrical vessel with curved base; motor and agitator; sterile air sparger at the bottom; foam breaker; temperature control jacket; pH control; sampling port; culture broth; exit gas (3). (b) Separation and purification of the product, formulation with preservatives, clinical trials for drugs (1). Strict quality control ensures each product is safe, pure and effective; procedures vary by product (1).

10. Q10 · 3 marks · Short answerHOTS / competency

    Suppose you cut a 10 kb circular plasmid with EcoRI and get fragments of 6 kb and 4 kb, and with BamH I you get a single 10 kb band. When cut with both, you get 6 kb, 3 kb and 1 kb fragments. How many sites does each enzyme have, and where is the BamH I site relative to the 4 kb EcoRI fragment? (3 marks)

    Hint

    Circular: n cuts give n fragments.

    Answer

    EcoRI gives 2 fragments from a circle, so it has 2 sites; BamH I gives 1 fragment (the opened 10 kb circle), so it has 1 site (1). In the double digest, the 6 kb fragment is unchanged but the 4 kb fragment has become 3 kb + 1 kb (1). So the BamH I site lies inside the 4 kb EcoRI fragment, 1 kb from one EcoRI site and 3 kb from the other. Check: 6 + 3 + 1 = 10 kb (1).

11. Q11 · 2 marks · Very short answerHOTS / competency

    Explain why traditional breeding may pass on undesirable genes, while genetic engineering does not. (2 marks)

    Hint

    Whole genome versus single gene.

    Answer

    Hybridisation mixes whole genomes, so undesirable genes are often included and multiplied along with the desired ones (1). Genetic engineering isolates and introduces only one or a set of desirable genes into the target organism (1).

12. Q12 · 2 marks · Very short answerHarder board level

    Why is a plasmid with a high copy number preferred when many copies of a gene are needed? Which feature of the vector decides this? (2 marks)

    Hint

    ori.

    Answer

    The linked gene is multiplied to the copy number of the plasmid, so a high-copy plasmid gives more copies of the gene per cell (1). The origin of replication controls the copy number, so a vector whose ori supports high copy number is chosen (1).

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. 1.Who made the first recombinant DNA, and when?Show answer

   Stanley Cohen and Herbert Boyer, in 1972.

2. 2.From which organism's plasmid was the antibiotic resistance gene taken?Show answer

   Salmonella typhimurium.

3. 3.What does 'R' in EcoRI stand for?Show answer

   The strain, RY 13.

4. 4.How many restriction enzymes are known now?Show answer

   More than 900, from over 230 strains of bacteria.

5. 5.What is agarose obtained from?Show answer

   Seaweeds.

6. 6.What is elution?Show answer

   Cutting out a DNA band from the agarose gel and extracting the DNA from it.

7. 7.What does rop in pBR322 code for?Show answer

   Proteins involved in the replication of the plasmid.

8. 8.What is T-DNA?Show answer

   The piece of DNA delivered by Agrobacterium tumefaciens that transforms plant cells into tumour cells.

9. 9.At what temperature is the heat shock given?Show answer

   42 °C.

10. 10.Name the source of the thermostable DNA polymerase used in PCR.Show answer

    The bacterium Thermus aquaticus.

11. 11.What volume of culture can bioreactors process?Show answer

    Large volumes, about 100–1000 litres.

12. 12.What is a recombinant protein?Show answer

    A protein made when a protein-coding gene is expressed in a heterologous host.

Quick revision checklist

The night before the exam: can you do each of these without looking?

• ☐Can you state the EFB definition and the two core techniques of biotechnology?
• ☐Can you describe how Cohen and Boyer made the first recombinant DNA?
• ☐Can you name the parts of 'EcoRI' and show its sticky ends?
• ☐Can you explain gel electrophoresis, staining and elution?
• ☐Can you label pBR322 and explain selection by antibiotic insertional inactivation and by blue-white screening?
• ☐Can you describe how E. coli is made competent and three other gene-transfer methods?
• ☐Can you write the three PCR steps and calculate copies after n cycles?
• ☐Can you label a stirred-tank bioreactor and list the steps of downstream processing?

Biotechnology: Principles and Processes: frequently asked questions