CBSE Class 12 Biology Biotechnology : Principles and Processes Worksheet

Read and download free pdf of CBSE Class 12 Biology Biotechnology : Principles and Processes Worksheet. Students and teachers of Class 12 Biology can get free printable Worksheets for Class 12 Biology in PDF format prepared as per the latest syllabus and examination pattern in your schools. Standard 12 students should practice questions and answers given here for Biology in Grade 12 which will help them to improve your knowledge of all important chapters and its topics. Students should also download free pdf of Class 12 Biology Worksheets prepared by school teachers as per the latest NCERT, CBSE, KVS books and syllabus issued this academic year and solve important problems with solutions on daily basis to get more score in school exams and tests

Worksheet for Class 12 Biology Chapter 11 Biotechnology Principles and Processes

Class 12 Biology students should refer to the following printable worksheet in Pdf for Chapter 11 Biotechnology Principles and Processes in standard 12. This test paper with questions and answers for Grade 12 Biology will be very useful for exams and help you to score good marks

Class 12 Biology Worksheet for Chapter 11 Biotechnology Principles and Processes

 

Important Questions for NCERT Class 12 Biology Biotechnology: Principles and Processes

Question. The specific DNA sequence in a chromosome which is responsible for initiation of replication is :
(a) Cloning region
(b) Termination region
(c) Initiation region
(d) Origin of replication

Answer : D

Question. Which of the following reproduction preserves the genetic informations ?
(a) Asexual reproduction
(b) Sexual reproduction
(c) Both (a) and (b)
(d) None of these

Answer : A

Question. The stickiness of the ends, facilitates the action of enzyme :
(a) DNA ligase
(b) DNA polymerase
(c) Alkaline phosphatase
(d) All of the above

Answer : A

Question. Two enzymes responsible for restricting the growth of bacteriophage in E.coli were isolated in 1963, one of these cut DNA, while other :
(a) Add propyl group to DNA
(b) Add ethyl group to DNA
(c) Add methyl group to DNA
(d) None of the above

Answer : C

Question. Taq polymerase is used in, polymerase chain reaction, because :
(a) It becomes inactive at high temperature
(b) it makes other enzyme active at high temperature
(c) It remains active during high temperature
(d) It is obtained from thermostable virus.

Answer : C

Question. The vessels, where large volumes of culture can be processed are :
(a) Bioreactors
(b) Biovessels
(c) Biocontainers
(d) All of above

Answer : A

Question. Bombardment of high velocity micro-particles of gold or tungsten coated with DNA on target cells is :
(a) Biolistics
(b) Micro-injection
(c) Electroporation
(d) Bombing

Answer : A

Question. In micro injection :
(a) DNA is bombarded on target cells
(b) DNA is placed through a vector
(c) DNA is directly injected into the nucleus of animal cell
(d) None of the above

Answer : C

Question. pBR322 has two antibiotic resistance genes, they are :
(a) Streptomycin and Ampicillin resistant gene
(b) Chloromycetin and tetracycline resistant gene
(c) Tetracycline and neomycin resistant genes
(d) Ampicillin and tetracyclin resistant genes

Answer : D

Question. Which one of the following is not required in PCR?
(a) Oligonucleotide primer
(b) DNA template
(c) Taq polymerase
(d) Helicase enzyme

Answer : D

Question. Select incorrect statement :
(a) Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as Lepidopterans, Coleopterans and Dipterans 
(b) RNA interference takes place in all eukaryotic organisms as a method of cellular defence
(c) Genetically modified crops are more sensitive to abiotic stresses
(d) Golden rice is protein enriched rice
(e) Agrobacterium is used to deliver desirable genes into animal cell
(1) only a
(2) a, b and c
(3) a, c and d
(4) c, d and e

Answer : D

Question. Most common matrix is agarose a natural polymer used in gel electrophoresis is extracted from :
(a) an animal
(b) a fungus
(c) Sea weeds
(d) None of these

Answer : C

Question. To isolate DNA from the plant cells we have to break the wall this is done by :
(a) Lysozyme
(b) Cellulase
(c) Chitinase
(d) Invertase

Answer : B

Question. Agrobacterium tumifaciens a pathogen transform normal plant cells into a tumor, similarly in animals the normal cells transformed into cancerous cells by:
(a) Retro viruses
(b) DNA viruses
(c) Ribo viruses
(d) None of these

Answer : A

Question. You have three copies of a particular DNA molecule what technique would you use to make more copies of the molecule?
(a) Gel electrophoresis
(b) Sequencing
(c) PCR
(d) Restriction fragment analysis

Answer : C

Question. Which of the following is best way to determine paternity ?
(a) Gene counting
(b) Chromosome counting
(c) DNA finger printing
(d) Protein analysis

Answer : C

Question. Restriction enzymes belong to a larger class of enzymes called :
(a) Cellulases
(b) Hydrolases
(c) Polymerases
(d) Nucleases

Answer : D

Question. Which one is not a basic step in genetically modifying an organism
(a) Identification of DNA with desirable genes
(b) Introduction of the identified DNA into the host
(c) Introduction of unidentified DNA into the host
(d) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Answer : C

Question. If any protein encoding gene is expressed in a hetero logous host then protein is known as :
(a) Recombinant gene
(b) Recombinant protein
(c) Selectable marker
(d) Homogenous protein

Answer : B

Question. Which enzyme is used in PCR technique ?
(a) Thermostable DNA polymerase
(b) Thermostable RNA polymerase
(c) Thermostable ligase
(d) Thermostable vector

Answer : A

Question. Which enzyme is used to isolate DNA from a plant cell?
(a) Chitinase
(b) DNA Polymerase
(c) Cellulase
(d) Amylase.

Answer: C

Question. A device in which large volume of living cells are cultured in order to get a specific product is called
(a) PCR
(b) agitator
(c) bioreactor
(d) assimilator

Answer: C

Question. Biolistic (gene gun) is suitable for
(a) disarming pathogen vectors
(b) transformation of plant cell
(c) constructing recombinant DNA by joining with vectors
(d) DNA fingerprinting

Answer: B

Question. Which is a natural genetic engineer of the plants?
(a) E. coli
(b) Agrobacterium tumefaciens
(c) Rhizobium
(d) Pseudomonas

Answer: B

Question. Which of the following gene helps in identifying transformed cells?
(a) plasmid
(b) selectable marker
(c) structural gene
(d) vector

Answer: B

Question. A tumor inducing plasmid widely used in the production of transgenic plants is that of
(a) Escherichia coli
(b) Bacillus thuringiensis
(c) Staphylococcus aureus
(d) Agrobacterium tumefaciens.

Answer: D

Question. In the screening process during rDNA experiments, clones that metabolize β-gal turn:
(a) Colourless
(b) Blue
(c) Yellow
(d) Green

Answer: B

Question. Biolistic method makes use of microparticles coated with DNA bombarded at cells to be transformed. These particles are made up of
(a) Zinc or tungsten
(b) Silicon or gold
(c) Tungsten or gold
(d) Selenium or Platinum

Answer: C

Question. Which of the following is a cloning vector?
(a) DNA of Salmonella typhimurium
(b) Ti plasmid
(c) Amp' and Tet' loci
(d) Ori minus PBR322

Answer: B

Question. The purified DNA is precipitated by adding
(a) Deoxyribonuclease
(b) Chilled ethanol
(c) Acetone
(d) Ethidium Bromide.

Answer: B

Question. Which of the following is the basic requirement of PCR reaction?
(a) Two oligonucleotide primers
(b) DNA segment to be amplified
(c) A heat-stable DNA polymerase
(d) All of the above

Answer: D

Question. A selectable marker is used to:
(a) help in eliminating the non-transformants so that the transformants can be regenerated.
(b) Identify the gene for a desired trait in an alien organism.
(c) Select a suitable vector for transformation in a specific crop.
(d) mark a gene on a chromosome for isolation using restriction enzyme.

Answer: C

Question. The PCR technique was developed by_________.
(a) Kohler
(b) Altman
(c) Milstein
(d) Kary Mullis

Answer: D

Question. The most important feature in a plasmid to be used as a vector is:
(a) Origin of replication (ori)
(b) Presence of a selectable marker
(c) Presence of sites for restriction endonuclease
(d) Its size

Answer: A

Question. Which of the following is incorrectly matched?
(a) RNA-Ribonuclease
(b) Proteins –Protease
(c) Fungus-Chitinase
(d) Plants –Lysozyme

Answer: D

Question. How the plasmid clones can be screened?
(a) By selectable markers
(b) By bacterial resistance gene
(c) For restriction site
(d) By ARS sequence

Answer: A

Question. Which of the following steps are catalysed by Taq polymerase in a PCR reaction?
(a) Denaturation of template DNA
(b) Annealing of primers to template DNA
(c) Extension of primer end on the template DNA
(d) All of the above

Answer: C

Question. Restriction endonuclease - Hind II always cuts DNA molecules at a particular point by recognizing a specific sequence of
(a) six base pairs
(b) five base pairs
(c) four base pairs
(d) seven base pairs

Answer: A 

Question. Which is not a component of the vector pBR322?
(a) Chloramphenicol selectable marker
(b) Replication proteins
(c) Origin of replication
(d) Cloning site for BamHI enzyme

Answer: B

Question. What is the process of binding of primer to the denatured strand called?
(a) Annealing
(b) Renaturation
(c) Denaturation
(d) None of the above

Answer: A

Question. Plants in comparison to animals are more rapidly manipulated by genetic engineering. Select out the most probable reason for this.
(a) Totipotency shown by plant cells.
(b) Single somatic cell can regenerate a whole plant body.
(c) Genetic engineering is supplemented with plant tissue culture techniques.
(d) All of the above

Answer: A

Question. The colonies of recombinant bacteria appear white in contrast to blue colonies of non-recombinant bacteria because of
(a) Non-recombinant bacteria containing β galactosidase
(b) Insertional inactivation of α-galactosidase in non-recombinant bacteria
(c) Insertional inactivation of β-galactosidase in recombinant bacteria
(d) Inactivation of glycosidase enzyme in recombinant bacteria

Answer: C

Question. Antibiotics are used in genetic engineering. They are useful
(a) to keep culture free of microbial infections
(b) to select healthy vectors
(c) to identify replication start sites
(d) as selectable markers

Answer: D

Question. While isolating DNA from bacteria, which of the following enzyme is not used?
(a) Deoxyribonuclease
(b) Lysozyme
(c) Ribonuclease
(d) Protease.

Answer: A

Question. Which of the following statements are true regarding PCR?
(a) Primer extension occurs at 72°C
(b) Denaturation involves heating at 90 to 98°C
(c) Annealing involves the binding of primer between 40 to 60C°C
(d) All of the above

Answer: D

Question. The construction of the first recombinant DNA was done by ?
(a) Stanley cohen and Herbert Boyer
(b) Nathan's and Smith
(c) Maeselson and Stahl
(d) Allec Jeffreys

Answer : A

Question. The most commonly used bioreactors are of
(a) Simple stirring type
(b) Sparged stirring type
(c) Both (a) and (b)
(d) None of the above

Answer : C

Question. Downstream processing is :
(a) Process of separation of DNA fragments
(b) Process of joining the vector and the host DNA
(c) Process including separation and purification of the product
(d) Process of transferring DNA.

Answer : C

Question. Vector which is commonly used to transfer foreign gene in a crop plant is :
(a) Plasmids of Salmonella
(b) l bacterio phage vector
(c) Ti plasmid of Agrobacterium tumifaciens
(d) None of the above

Answer : C

Question. Which of the following enzymes is known as 'genetic glue'?
(a) DNA polymerase
(b) Alkaline phosphatase
(c) DNA ligase
(d) All of the above

Answer : C

Assertion - Reason Questions:

In the following questions, a statement of assertion is followed by a statement of reason. Mark the correct choice as:
(a) If both Assertion and Reason are true and Reason is the correct explanation of Assertion.
(b) If both Assertion and Reason are true but Reason is not the correct explanation of Assertion.
(c) If Assertion is true but Reason is false.
(d) If both Assertion and Reason are false.

Question. Assertion: Cloning vector should have selectable marker.
Reason: Selectable marker helps in identifying and eliminating non – transformants and selectively permitting the growth of transformants.

Answer: A

Question. Assertion: the double-stranded DNA is denatured by subjecting it to high temperature of 940 C for 15 seconds.
Reason: One of the separated strands of the DNA is destroyed during denaturation and the other act as a template.

Answer: C

Question. Assertion: It is essential to have few cloning sites in a cloning vector.
Reason: It helps in identifying and eliminating non – transformants and selectively permitting the growth of transformants.

Answer: C

Question. Assertion: In a bioreactor, temperature, pH, substrate, salts, vitamins, and oxygen are maintained.
Reason: They are added to maintain the proper growth of the organism and to achieve desired product optimally.

Answer: A

Question. Assertion: Foreign DNA and vector DNA are cut with the help of ligase.
Reason: Ligase act on sugar phosphate backbone of DNA.

Answer: D

Question. Assertion: In gel electrophoresis, DNA fragments are separated.
Reason: DNA is negatively charged, so it moves towards anode under electric field.

Answer: A

Question. Assertion: The genes encoding resistance to antibiotics are considered as useful selectable markers for E. coli
Reason: The normal E. coli does not carry resistance against any of the antibiotics.

Answer: C

Question. Assertion: Agrobacterium tumefaciens is popular in genetic engineering because this bacterium is associated with the roots of all cereal and pulse crops.
Reason: A gene incorporated in the bacterial chromosomal genome, gets automatically transferred to the crop with which the bacterium is associated.

Answer: A

Question. Assertion: The recombinant cells are multiplied in a continuous culture system
Reason: It is to maintain the cells in their physiologically most active log/exponential phase.

Answer: A

Question. Assertion: Chitinase is used to isolate DNA from bread Mould
Reason: Chitinase is an enzyme that degrades chitin, which is a chief constituent of fungal cell walls.

Answer: A

Question. Assertion: The uptake of DNA during transformation is an active, energy requiring process.
Reason: Transformation occurs in only those bacteria, which possess the enzymatic machinery involved in the active uptake and recombination.

Answer: C

Question. Assertion: Bioreactors are used for the large-scale production of the desired products.
Reason: Bioreactor designing only needs complete information of biological systems.

Answer: B

Question. Assertion: In the annealing step of PCR, primers are added at free 3‘ end of the target DNA and are catalyzed at a temperature of 60-500 C.
Reason: The extension step is catalysed by Taq polymerase enzyme at a temperature of 720 C.

Answer: B

Question. Assertion: E. coli having pBR322 with DNA insert at Bam HI site cannot grow in medium containing tetracycline.
Reason: Recognition site for Bam HI is present in tetR region of pBR32.

Answer: B

Question. Assertion: Selectable markers helps in identifying and eliminating nontransformants and selectively permitting the growth of transformants
Reason: If a recombinant DNA bearing gene for resistance to ampicillin is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells.

Answer: B

Very Short Answer Type Questions

Question. What is the significance of adding proteases at the time of isolation of genetic material (DNA)?
Answer : Proteases degrade the proteins present inside a cell (from which DNA is being isolated). If the proteins are not removed from DNA preparation then they could interfere with any downstream treatment of DNA.

Question. While doing a PCR, ‘denaturation’ step is missed. What will be its effect on the process?
Answer : If denaturation of double-stranded DNA does not take place then primers will not be able to anneal (joining) to the template. Hence, no extension will take place and after are there will be no amplification.

Question. How is copy number of the plasmid vector related to yield of recombinant protein?
Answer : The recombinant DNA can multiply as many times as the copy number of the vector plasmid thereby determining the yield of recombinant protein. So, higher the copy number of plasmid vector, higher the copy number of gene and consequently, protein coded by the gene is produced in high amount.

Question. Would you choose an exonuclease, while producing a recombinant DNA molecule?
Answer : No, as exonuclease acts on the free ends of linear DNA molecule. Therefore, instead of producing DNA fragments with sticky ends, it will shorten or completely degrade the DNA fragment containing the gene of interest and the circular plasmid (vector) will not get cut as it lacks free ends.

Question. Name a recombinant vaccine that is currently being used in vaccination program.
Answer : Hepatitis-B recombinant vaccine (engerix) is used for vaccination of hapatitis virus.

Short Answer Type Questions

Question. Both a wine maker and a molecular biologist who had developed a recombinant vaccine claim to be biotechnologists. Who in your opinion is correct?
Answer : In my opinion both of them are correct. As biotechnology is a very wide area which deals with techniques of using a ‘natural’ organism (or its parts) as well as genetically modified organism to produce and processes useful for mankind. A wine maker employs a strain of yeast to produce wine by fermentation (a natural phenomenon), while the molecular biologist has cloned gene for the antigen (that is used as vaccine) in an organism which allows the production of the antigen in large amount.

Question. How does one visualise DNA on an agarose gel?
Answer : A compound called ethidium bromide stains DNA, which on exposure with ultra-violet, (uv) radiation gives orange light band of DNA. Hence, DNA fragments appear as orange band in the presence of ethidium bromide and UV light.

Question. A plasmid without a selectable marker was choosen as vector for cloning a gene. How does this affect the experiment?
Answer : In a gene cloning experiment, first a recombinant DNA molecule is constructed, where the gene of interest is ligated to the vector (the step would not be affected) and introduced inside the host cell (transformation). Since, not all the cells get transformed with the recombinant/plasmid DNA, in the absence of selectable marker, it will be difficult to distinguish between transformants and non-transformant, because role of selectable marker is in the selection of transformants.

Question. Identify and explain A, B and C in the PCR diagram given below.
CBSE Class 12 Biology Biotechnology Principles and Processes Worksheet

Answer : Region to be amplified 
CBSE Class 12 Biology Biotechnology Principles and Processes Worksheet
 In PCR, each cycle has three steps (i) Denaturation of DNA Sample Unwinding of two strand of DNA by heating the sample at 92-94°C.
(ii) Primer Annealing Primers get positioned on the exposed nucleotides as per base pairing rules.
(iii) Extension of Primers DNA polymerase recognises primers as ‘start’ tags and begins to extend the primers using the free nucleotides provided in the reaction and the genomic DNA as template. With each round of reactions, the DNA doubles.

Question. Name the regions marked A, B and C.
CBSE Class 12 Biology Biotechnology Principles and Processes Worksheet
Answer : Region A Bam HI Region B Pot I Region C ampR. E. coli cloning vector pBR322 showing restriction sites (Hind III, Eco RI, Bam HI, Sal I, Pvu II, Pst I, Cla I), Ori and antibiotic resistance genes (amp and tet ) R R . Rop codes for the proteins involved in the replication of the plasmid. 

CBSE Class 12 Biology Biotechnology Principles and Processes Worksheet_3

Question. A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason?
Answer : The reasons are as follows (i) DNA sample that was loaded on the gel may have got contaminated with nuclease (exo or endo both) and completely degraded. (ii) Electrodes were put in opposite orientation in the gel assembly that is anode towards the wells (where DNA sample is loaded). Since, DNA molecules are negatively charged, they move towards anode and hence, move out of the gel instead of moving into the matrix of gel. (iii) Ethidium bromide was not added at all or was not added in sufficient concentration and so DNA was not visible.
 

Long Answer Type Questions:

Question. For selection of recombinants, insertional inactivation of antibiotic marker has been supercoded by insertional inactivation of a marker gene coding for a chromogenic substrate. Give reasons.
Answer : In selection of recombinants due to inactivation of antibiotics, the transformed cells are first plated on the antibiotic plate which has not been insertionally inactivated (i.e., ampicillin) and incubated overnight for growth of transformants. For selection of recombinants, these transformants are replica-plated on second antibiotic (say, tetracycline) plate (which got inactivated due to insertion of gene). Non-recombinants grow on both the plates (one carrying ampicillin and the other carrying tetracycline) while recombinants will grow only on ampicillin plate.This entire exercise is labourious and takes more time (two overnight incubation) as well. However, if we choose insertional inactivation of a marker that produces colour in the presence of a chromogenic compound, we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth.

Question. Describe the role of Agrobacterium tumefaciens in transforming a plant cell.
Answer : A soil-inhabiting, plant pathogenic bacterium, Agrobacterium tumefaciens, infects broad-leaved crops including tomato, soyabean, sunflower and cotton, but not the cereals. It causes tumours called crown galls. Tumour formation is induced by its plasmid, which is, therefore called Ti-plasmid (Ti for tumour inducing). The Ti-plasmid integrates a segment of its DNA, termed T-DNA, into the chromosomal DNA of its host plant cells. The T-DNA causes tumours. As gene transfer occurs without human effort, the bacterium is known as natural genetic engineer of plants. Plant molecular biologists have started using Ti-plasmids as vectors to transfer foreign genes of interest into the target plant cells. They use a version of the plasmid from which tumour forming gene has been eliminated. The transformed bacteria do not cause disease, but still deliver genes of interest into a variety of plants.

Question. Illustrate the design of a bioreactor. Highlight the difference between a flask in your laboratory and a bioreactor which allows cells to grow in a continuous culture system.
Answer : Bioreactors are vessels of large volumes (100-1000 L) in which raw materials are biologically converted into specific products. The most commonly used bioreactors are of stirring type, which are shown in figure. (Image 209) A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor. If you look at the figure closely you will see that the bioreactor has an agitator system, an oxygen delivery system and a form control system, a temperature control system, pH control system and sampling ports so, that small volumes of the culture can be withdrawn periodically. Small volume cultures are usually employed in laboratories in a flask for research and production of less quantities of products. However, large scale production of the products is carried out in bioreactors.

Question. a) Why are engineered vectors preferred by biotechnologists for transferring the desired genes into another organism?
(b) Explain how do ―ori‖ selectable markers‖ and‖ cloning sites‖ facilitate cloning into a vector?
Answer: (a)Engineered vectors are preferred because they help easy linking of foreign DNA and selection of recombinants from non-recombinants.
(b) (i) Origin of replication (Ori)
-It is the sequence of DNA from where replication starts.
-Any piece of alien DNA linked to it is made to replicate within the host cell
-It also decides the copy number of linked DNA
(ii) Selectable marker
-The selectable marker is a gene in the vector, which helps in selecting the transformant/recombinant cells from the non-recombinants. Eg. the genes encoding resistance to antibiotics.
(iii) Cloning sites
-The vector should have very few, preferably single recognition sites to link the alien/foreign DNA.

Question. What is downstream processing? Describe the steps in downstream processing.
Answer: The process of formulation, separation and purification of rDNA products made in Bioreactors is called downstream processing.
Steps in downstream processing.
i)Separation of biomass: the (microbial cells are separated from the culture medium. If the product is biomass then it is recovered for processing and spent medium is discarded. Cell mass is separated from the fermented broth by centrifugation or ultracentrifugation. When there is no aeration and agitation some of the microbial cells settle down in the fermentor.
i) Cell disruption: if desired product is intracellular the cell biomass can be disrupted so that the product is released.
iii) concentration of broth: The spent medium is concentrated if the product is extracellular.
iv) Initial purification of metabolites: methods for recovery of product from the clarified fermented broth -precipitation, solvent extraction, ultrafiltration, ion exchange chromatography, adsorption and solvent extraction.
v) Metabolite specific purification: Specific purification methods are used when desired metabolite is purified to a very high degree.
vi) De watering: When a low amount of product is found in very large volume of spent medium, volume is reduced by removing water to concentrate the product.it is done by vaccum drying or reverse osmosis.
vii) Polishing of metabolites: Final step of making the product to 98%-100% pure.
Purified product is mixed with excipients. The formulated product is packed and sent to the market for the consumers.

Question. With the help of a neat labelled sketch, explain the formation of recombinant DNA by action of restriction endonuclease enzyme EcoR1.
Answer: DIAGRAM –Refer Page 196 Fig 11.1NCERT text
Restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA.
-The recognition site for EcoRI - 5' - G A A T T C - 3'
3' - C T T A A G - 5'
-Restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites, but between the same two bases on the opposite strands:
-The enzyme cuts both the vector DNA and the foreign DNA at the same site.
EcoRI cuts the DNA between bases G and A only when the sequence GAATTC is present in the DNA.
-This leaves single-stranded portions at the end which are overhanging stretches called sticky ends.
-sticky end form hydrogen bonds with their complementary cut counterparts. This stickiness of the end facilitates the action of enzyme DNA ligase.
-Thus, Recombinant DNA is formed.

Question. Any recombinant DNA with a desired gene is required in billion copies for commercial use. How is the amplification done? Explain.
Answer: Amplification of gene is done using polymerase Chain Reaction (PCR). it is carried out in the following steps:
(i) Denaturation The double stranded DNA is denatured by applying high temperature of 95°C for 15 seconds. Each separated strand acts as a template.
(ii) Annealing Two sets of primers are added, which anneal to the 3′ end of each separated strand.
(iii) Extension DNA polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction. Taq polymerase is used in the reaction, which can tolerate heat. All these steps are repeated many times to get several copies of the desired DNA.

Question. How is the bacterium Thermus aquaticus employed in recombinant DNA technology?
Answer: Thermus aquaticus, a bacterium yields DNA polymerase used in PCR in recombinant DNA technology.
(i) This enzyme remains active during the high temperature applied in the denaturation of double stranded DNA.
(ii) It extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
(iii) Repeated amplification is achieved by this enzyme. The amplified fragments, if desired can be used to ligate with a vector for further cloning.

Question. Draw well labelled diagrams of simple stirred tank bioreactor and sparged stirred tank bioreactor. distinguish between simple stirred tank bioreactor and sparged stirred tank bioreactor.
Answer: i) There is increased surface area for oxygen transfer in the Sparged Stirred bio reactor, whereas there is less surface area as compared to Sparged tank for oxygen area in Simple Stirred bio reactors.
ii) Bubbles increase the oxygen transfer area in Sparged whereas there is absence of oxygen bubbles in Simple stirred

Question. What is bioreactor? Draw a labelled diagram of a sparged stirred bioreactor. Explain its functioning.
Answer: Bioreactors are vessels in which raw materials are biologically converted into specific products using microbial, plant or animal cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions like temperature, pH, substrate, salts, vitamins and oxygen. In a sparged stirred-tank bioreactor, the sterile air is sparged through the reactor.
Sparged stirred-tank bioreactor has increased surface area for oxygen transfer than simple stirred-tank. 

Question. With the help of diagrams enumerate the different steps in recombinant DNA technology.
Answer:
• Isolation of genetic material which has the gene of interest.
• Cutting of gene of interest from genome and vector with the same restriction endonuclease enzyme.
• Amplifying gene of interest (PCR).
• Ligating gene of interest and vector using DNA ligase forming rDNA.
• Transformation of rDNA into the host cell.
• Multiplying host cell to create clones.
• Figure 11.2 Diagrammatic representation of recombinant DNA technology

Question. Describe the parts of a Simple stirred-tank bioreactor.
Answer: 
i) An agitator system
ii) oxygen delivery system
iii) Foam control system
iv) temperature control system
v) pH control system
vi) Sampling ports
vii) Cleaning and sterilization system.
viii) A sump and dump line for emptying of the reactor.

Question. Sequentially state the process you would adopt for getting a recombinant protein?
Answer: 
i) Isolation of DNA (enzymes used), cutting the DNA,
ii) Separation of fragments,
iii) PCR,
iv) introducing into host cell,
v) obtaining gene product in the Bioreactor,
vi) downstream processing

Question. List the steps involved in rDNA technology?
Answer: 
• Isolation of a desired DNA fragment.
• Ligation of the DNA fragment into a vector.
• Transferring the recombinant DNA into the host.
• Culturing the host cells in a medium at large scale and extraction of the desired product.


1 Name an enzyme catalysing the removal of nucleotides from the ends of DNA.

2 Write the Significance of 'heat shock' method in bacterial transformation.

3 Name two bacteria which are the sources of restriction endonuclease?

4 Identify the steps of PCR in which Taq polymerase is used.

5 Define recombinant protein.

6 What does ‘competent’ refer to in competent cells used in transformation experiments? Describe the role of CaCl2 in the preparation of competent cells?

7 What is the significance of adding proteases at the time of isolation of genetic material (DNA). Name the enzyme used to digest the cell wall of fungi.

8 What modification is done on the Ti plasmid of Agrobacterium tumefaciens to convert it into a cloning vector? Name the vector used to transfer gene of interest to animal cell.

9 How does one visualise DNA on an agarose gel?

10 For selection of recombinants, insertional inactivation of antibiotic marker has been superceded by insertional inactivation of a marker gene coding for a chormogenic substrate. Give reasons.

11 Describe the role of Agrobacterium tumefaciens in transforming a plant cell.

12 a) What are molecular scissors? Give one example. b) Explain their role in recombinant DNA technology.

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