Class 12 Biology Chapter 9 Important Questions – Biotechnology: Principles and Processes

Biotechnology uses organisms, cells or enzymes to produce useful products and processes.

This chapter explains genetic engineering, recombinant DNA tools, PCR, cloning vectors, bioreactors and downstream processing.

Class 12 Biology Chapter 9 Important Questions help students revise the tools and stages used in modern biotechnology. The chapter explains how a selected gene is isolated, copied, inserted into a vector and introduced into a suitable host.

Students should understand the purpose of each enzyme and vector component. They should also learn the correct sequence of recombinant DNA technology, from DNA isolation to product purification.

Key Takeaways

• Genetic engineering changes genetic material and introduces it into a host.
• Bioprocess engineering supports large-scale production under sterile conditions.
• Restriction endonucleases cut DNA at specific recognition sequences.
• DNA ligase joins compatible DNA fragments.
• Plasmids and bacteriophages can act as cloning vectors.
• A vector requires an origin of replication, selectable marker and cloning site.
• PCR produces numerous copies of a selected DNA segment.
• Bioreactors maintain suitable conditions for large-scale production.
• Downstream processing includes product separation, purification and formulation.

Important Terms from Biotechnology: Principles and Processes

Main Steps of Recombinant DNA Technology

1. Isolation of genetic material
2. Cutting DNA at specific locations
3. Separation of DNA fragments
4. Amplification of the desired gene
5. Ligation into a suitable vector
6. Introduction into a host cell
7. Selection of transformed cells
8. Expression of the foreign gene
9. Large-scale production
10. Downstream processing

Access Class 12 Biology Chapter 9 Important Questions in 30 Minutes

First 10 minutes: Revise biotechnology principles, restriction enzymes and gel electrophoresis.

Next 10 minutes: Review cloning vectors, selectable markers and gene-transfer methods.

Final 10 minutes: Practise PCR, recombinant DNA steps, bioreactors and downstream processing.

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Important Questions for Class 12 Biology Chapter 9

Q1. What is biotechnology? State its two core techniques.

Answer:

Biotechnology uses living organisms, cells, enzymes or their components to produce useful products and processes.

The two core techniques of modern biotechnology are:

Genetic engineering: It involves altering DNA or RNA and introducing it into a host organism.

Bioprocess engineering: It maintains sterile conditions for growing selected cells or microbes on a large scale.

Genetic engineering creates the desired biological system. Bioprocess engineering supports its commercial production.

Q2. State the three basic steps involved in genetically modifying an organism.

Answer:

The three basic steps are:

1. Identification of DNA containing the desirable gene
2. Introduction of the identified DNA into a suitable host
3. Maintenance of the introduced DNA in the host and its transfer to progeny

The foreign DNA must replicate inside the host to remain present in future cells.

Q3. How was the first artificial recombinant DNA molecule constructed?

Answer:

Stanley Cohen and Herbert Boyer constructed an artificial recombinant DNA molecule in 1972.

They isolated an antibiotic-resistance gene from a plasmid. A restriction enzyme was used to cut the desired gene and a plasmid vector.

The gene was joined to the plasmid using DNA ligase. This recombinant plasmid was then introduced into Escherichia coli.

The bacteria replicated the plasmid and produced multiple copies of the antibiotic-resistance gene.

Q4. Differentiate between exonucleases and endonucleases.

Answer:

Restriction endonucleases are called molecular scissors because they cut DNA at specific internal locations.

Q5. Explain the naming of the restriction enzyme EcoRI.

Answer:

The name EcoRI is derived from the bacterium from which it was isolated.

• E comes from the genus Escherichia.
• co comes from the species coli.
• R represents the bacterial strain RY13.
• I indicates that it was the first restriction enzyme isolated from that strain.

Restriction enzymes isolated later from the same strain receive Roman numerals II, III and so on.

Q6. What is a palindromic DNA sequence? Explain using the EcoRI recognition sequence.

Answer:

A palindromic DNA sequence reads the same on both strands when both are read in the 5′ to 3′ direction.

The recognition sequence of EcoRI is:

5′–GAATTC–3′
3′–CTTAAG–5′

EcoRI cuts between G and A on both strands:

5′–G | AATTC–3′
3′–CTTAA | G–5′

This produces complementary single-stranded overhangs called sticky ends.

Q7. What are sticky ends? Why are they useful in recombinant DNA technology?

Answer:

Sticky ends are short, single-stranded DNA overhangs produced when restriction enzymes make staggered cuts.

They can form hydrogen bonds with complementary sticky ends produced by the same restriction enzyme.

When the source DNA and vector are cut using the same enzyme, their compatible ends can pair. DNA ligase then joins the sugar-phosphate backbones.

Sticky ends therefore make the formation of recombinant DNA more precise.

Q8. Explain the separation and isolation of DNA fragments through gel electrophoresis.

Answer:

Restriction digestion produces DNA fragments of different sizes.

These fragments are loaded into wells in an agarose gel. DNA carries a negative charge because of its phosphate groups, so it moves towards the positive electrode or anode.

Smaller fragments move more easily through the agarose matrix and travel farther than larger fragments.

The separated fragments are stained with ethidium bromide and viewed under ultraviolet light. They appear as bright orange bands.

The required band is cut from the gel and the DNA is extracted. This process is called elution.

Q9. State the essential features of a cloning vector.

Answer:

A suitable cloning vector should contain:

Origin of replication: It allows the vector and inserted DNA to replicate inside the host.

Selectable marker: It helps identify cells that have received the vector.

Cloning site: It provides a unique restriction enzyme recognition site for inserting foreign DNA.

A useful vector should also be small enough to isolate and manipulate easily.

Q10. What is the importance of the origin of replication in a cloning vector?

Answer:

The origin of replication, or ori, is the DNA sequence where replication begins.

Foreign DNA linked to the ori can replicate inside the host cell along with the vector.

The ori also influences the copy number of the vector. A vector with a high-copy-number origin produces many copies of the inserted gene.

Without an origin of replication, the foreign DNA may not multiply or pass to daughter cells.

Q11. What are selectable markers? How are antibiotic-resistance genes used as markers?

Answer:

Selectable markers help identify transformants and eliminate non-transformants.

Genes providing resistance to antibiotics such as ampicillin or tetracycline are commonly used in bacterial vectors.

After transformation, bacterial cells are grown on a medium containing the antibiotic.

• Cells without the vector die.
• Cells carrying the resistance gene survive.

The surviving colonies are identified as transformants. Additional screening may be needed to distinguish recombinants from non-recombinant transformants.

Q12. Explain insertional inactivation and blue-white selection.

Answer:

Insertional inactivation occurs when foreign DNA is inserted within the coding sequence of a marker gene and disrupts its function.

In blue-white selection, foreign DNA is inserted into the gene coding for beta-galactosidase.

When a chromogenic substrate is present:

• Non-recombinant bacteria produce functional beta-galactosidase and form blue colonies.
• Recombinant bacteria have an interrupted gene and form colourless or white colonies.

This method allows recombinants to be identified on a single plate.

Q13. How is the Ti plasmid used as a cloning vector in plants?

Answer:

Agrobacterium tumefaciens naturally transfers a section of its Ti plasmid, called T-DNA, into plant cells.

In nature, this DNA causes tumour formation. Scientists remove the disease-causing genes and insert a desired gene into the T-DNA region.

The modified Ti plasmid retains its ability to transfer DNA but no longer causes disease.

It is therefore used as a vector to introduce useful genes into plant cells.

Q14. Explain how bacterial cells are made competent to receive recombinant DNA.

Answer:

DNA cannot easily cross the bacterial cell membrane because it is hydrophilic.

Bacterial cells are treated with a suitable concentration of divalent calcium ions. This treatment increases membrane permeability and helps DNA enter through pores in the cell wall.

The cells are then:

1. Mixed with recombinant DNA on ice
2. Briefly exposed to approximately 42°C
3. Returned to ice

This heat-shock treatment enables bacteria to take up recombinant DNA.

Q15. Describe three methods used to introduce foreign DNA into host cells.

Answer:

Microinjection: Recombinant DNA is directly injected into the nucleus of an animal cell using a fine needle.

Biolistics or gene gun: Plant cells are bombarded with microscopic gold or tungsten particles coated with DNA.

Disarmed pathogen vectors: Modified viruses or bacteria transfer desired genes without causing their original disease.

Competent bacterial cells may also receive recombinant DNA through calcium treatment and heat shock.

Q16. Explain the three stages of PCR and the role of Taq polymerase.

Answer:

PCR stands for Polymerase Chain Reaction. It produces multiple copies of a selected DNA segment.

Denaturation: High temperature separates the two DNA strands.

Annealing: The temperature is lowered so primers bind to complementary sequences.

Extension: DNA polymerase adds nucleotides from the primer ends and produces new strands.

Taq polymerase is obtained from Thermus aquaticus. It remains active at the high temperatures used during repeated PCR cycles.

Repeated cycles can amplify the target DNA to approximately a billion copies.

Q17. Describe the steps used to isolate pure DNA from cells.

Answer:

Cells are first broken open to release DNA and other cellular components.

Different enzymes are used depending on the cell type:

• Lysozyme breaks bacterial cell walls.
• Cellulase breaks plant cell walls.
• Chitinase breaks fungal cell walls.

Ribonuclease removes RNA, while protease removes proteins.

Other molecules are removed through suitable treatments. Chilled ethanol is then added, causing purified DNA to precipitate as fine threads.

Q18. Describe the main steps of recombinant DNA technology.

Answer:

The process begins with the isolation of pure DNA from the source organism.

The source DNA and vector are cut using the same restriction enzyme. The desired DNA fragment is separated through gel electrophoresis and extracted by elution.

DNA ligase joins the gene of interest with the opened vector. The resulting recombinant DNA is introduced into a suitable host.

Transformants and recombinants are selected using markers. The host cells are cultured so the foreign gene can replicate and express the desired product.

Large-scale production takes place in bioreactors. The final product is then separated and purified through downstream processing.

Q19. What is a bioreactor? Explain the main features of a stirred-tank bioreactor.

Answer:

A bioreactor is a large vessel in which microbial, plant, animal or human cells convert raw materials into useful biological products.

A stirred-tank bioreactor usually has:

• An agitator for continuous mixing
• An oxygen-delivery system
• A temperature-control system
• A pH-control system
• A foam-control system
• Sampling ports
• Inlets for nutrients and other materials

The stirrer distributes nutrients and oxygen evenly. In a sparged stirred-tank reactor, sterile air is also bubbled through the culture.

Q20. What is downstream processing? Why is it necessary?

Answer:

Downstream processing includes the steps performed after the desired biological product has been produced.

It generally involves:

• Separation of the product from cells and culture medium
• Purification of the product
• Formulation with suitable preservatives
• Quality-control testing
• Clinical trials where required

These steps ensure that the final product is pure, stable, safe and suitable for marketing.

The exact process differs according to the product being manufactured.

How to Prepare Biotechnology: Principles and Processes

Begin by learning the purpose of each tool: restriction enzymes cut DNA, ligase joins DNA and vectors carry DNA. Link each tool with its exact role instead of memorising isolated definitions.

Draw the EcoRI recognition sequence, pBR322 vector, PCR cycle and stirred-tank bioreactor. Diagrams make the sequence of steps easier to remember.

Practise the complete recombinant DNA process in the correct order. Questions often test what happens before or after a particular stage.

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