CBSE Class 12 Biology Biotechnology Principles and Processes Worksheet Set B

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 : α-1 antitrypsin is
(a) an antacid.
(b) an enzyme
(c) used to treat arthritis
(d) used to treat emphysema 

Answer : D

Question : Stem cells can be obtained from
(a) from embryo
(b) from placenta
(c) from body parts
(d) from an embryo or tissues in the body. 

Answer : D

Question : C-peptide of human insulin is
(a) a part of mature insulin molecule.
(b) responsible for formation of disulphide ridges.
(c) removed during maturation of pro-insulin to insulin.
(d) responsible for its biological activity.

Answer : C

Question : A probe which is a molecule used to locate specific sequences in a mixture of DNA or RNA molecules could be
(a) a single stranded RNA
(b) a single stranded DNA
(c) either RNA or DNA
(d) can be ss-DNA but not ss RNA

Answer : C

Question : What triggers activation of protoxin to active Bt toxin of Bacillus thuringiensis in bollworm ?
(a) Body temperature
(b) Moist surface of midgut
(c) Alkaline pH of gut
(d) Acidic pH of stomach

Answer : C

Question : Golden rice is
(a) a variety of rice grown along the yellow river in China.
(b) long stored rice having yellow colour tint.
(c) a transgenic rice having gene for β-carotene.
(d) wild variety of rice with yellow coloured grains.

Answer : C

Question : The site of production of ADA in the body is
(a) bone marrow
(b) lymphocytes
(c) blood plasma
(d) monocytes

Answer : B

Question : The polio vaccine was tested on :
(a) Mice
(b) Monkey
(c) Rabbit
(d) Dog

Answer : A

Question : Bt cotton is not
(a) a genetically modified (GM) plant.
(b) insect resistant.
(c) a bacterial gene expressing system.
(d) resistant to all pests

Answer : D

Question : Which body of the Government of India regulates GM research and safety of introducing GM organisms for public services.
(a) Bio-safety committee
(b) Indian council of Agricultural Research
(c) Genetic Engineering Approval committee
(d) Research Committee on Genetic Manipulation.

Answer : C

Question : A pro-toxin is
(a) a primitive toxin
(b) a denatured toxin
(c) toxin produced by protozoa
(d) inactive toxin 

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.

Question. Why is it essential to have ‘selectable marker’ in cloning vector.
Answer : Selectable marker helps in the identification and elimination of non-transformants and permitting the growth of the transformants. Therefore, they are considered essential in cloning vector. 

Question. How is the action of normal endonuclease enzymes different from that of restriction endonuclease ?
Answer : Normal endonuclease cuts at random position within a DNA sequence, whereas restriction endonuclease recognizes and cut specific nucleotide sequences within DNA. 

Question. Name the host cells in which micro–injection technique is used to introduce an alien DNA.
Answer : Animal cell.

Question. Write the two components of the first artificial recombinant DNA molecule constructed by Cohen and Boyer. 
Answer : The two components are antibiotic resistant gene and plasmid vector of Salmonella typhimurium. 

Question. Name the technique that is used to alter the chemistry of genetic material (DNA, RNA) to obtain desired result.
Answer : Genetic Engineering / Biochemical Engineering / Biotechnology. 

Question. Name the material used as matrix in gel electrophoresis and mention its role.
Answer : Agarose is the most commonly used matrix in DNA gel electrophoresis. It provides sieving effect for separation of DNA fragments according to their size. 

Question. Name two enzymes that are essential for constructing a recombinant DNA.
Answer : Restriction enzymes / polymerase enzymes / ligase 

Question. State what happens when an alien gene is ligated at Pvu I site of pBR322 plasmid.
Answer : When an alien gene is ligated at the Pvu I site of ampicillin resistance gene in the vector pBR322, the recombinant plasmids lose ampicillin resistance due to insertion of the foreign DNA.

Question. How can following be made possible for biotechnology experiments?
(i) Isolation of DNA from bacterial cell.
(ii) Reintroduction of recombinant DNA into a bacterial cell.
Answer : (i) By treating the cell with the enzyme lysozyme.
(ii) By making the bacterial cell competent. 

Question. Mention the type of host cells suitable for the gene guns to introduce an alien DNA.
Answer : Plant cells. 

Question. Why EtBr is used in gel electrophoresis in spite of it being highly carcinogenic ?
Answer : EtBr is an intercalating agent. It stacks itself in the DNA bases, and fluoresce under U.V light, thus helps in identification of DNA.

Short Answer Type Questions

Question. Explain the role of Ti plasmid in biotechnology.
Answer : (i) The Ti plasmid (tumor-inducing plasmid) of Agrobacterium tumefaciens has been modified (does not cause tumour) and used as a cloning vector. The Ti plasmid integrates a segment of its DNA, termed T-DNA into the chromosomal DNA of its host plant cells.
(ii) The T-DNA plasmid causes tumours. As gene transfer occurs without human effort, the bacterium is known as ‘natural genetic engineer’ of plants. Ti plasmids as vectors, transfer foreign genes of interest into the target cells. 

Question. How does a restriction endonuclease help in DNA recombinant technology ?
OR
Explain the mode of action of EcoRI.
Answer : Restriction endonuclease (EcoRI) inspects length of DNA and recognises specific palindromic nucleotide sequence, binds with DNA, cuts each of the two strands of double helix at specific points.
This leaves the single stranded overhanging stretches at the ends. They are called sticky ends.
They form H-bonds with their complementary cut counterparts. This stickiness facilitates action of DNA ligase, when cut by the same restriction enzyme. The resultant DNA fragments have the same kind of sticky ends and these are joined together by DNA ligase. 

Question. Explain palindromic nucleotide sequence with the help of a suitable example.
Answer : Palindrome in DNA is a sequence of base pairs that reads the same on two strands when orientation of reading is the same.
Example : 5’ — GAATTC — 3’
3’—CTTAAG — 5’ 2

Question. Explain how to find whether E. coli bacterium has transformed or not, when, a recombinant DNA bearing ampicillin-resistant gene is transferred into it. 
Answer : The recombinant / transformant may be found out from non-recombinant / non-transformant by plating the transformants on ampicillin containing medium. The transformants growing on ampicillin containing medium are then transferred to tetracycline containing medium. The recombinant will grow on ampicillin containing medium but not on that containing tetracycline. But nonrecombinant will grow on both tetracycline and ampicillin containing media. 

Question. List the key tools used in recombinant DNA technology. 
Answer : Restriction enzymes / Polymerase enzymes / Ligase enzymes / Vectors / Host organisims / E. coli/ Agrobacterium. 

Question. How are ‘sticky ends’ formed on a DNA strand ? Why are they so called ? 
Answer : Restriction enzymes cut the strands of the DNA, a little away from the centre of the palindromic sites, but between the same two bases on opposite strands. 
These overhang stretches are called as sticky ends.
They form hydrogen bonds with their complementary cut counterparts. 

Question. Explain with the help of a suitable example the naming of a restriction endonuclease.
Answer : EcoRI.
The first letter of the name comes from the genus and the next two from the name of the species of the bacterium i.e. prokaryotic cell. Thus Eco stands for the genus and species of the prokaryotic cell from which the enzyme was isolated i.e. E. coli R stands for strain. ‘I’ follows order in which enzyme was isolated. 

Question. (i) Name the selectable markers in the cloning vector pBR322. Mention the role they play.
(ii) Why is the coding sequence of an enzyme β-galactosidase a preferred selectable marker in comparison to the ones named above ?
Answer : (i) amp^R / ampicillin resistance genes, tet^R/ tetracycline resistance gene. 
They help in identifying and eliminating nontransformants / non- recombinants and selectively permitting the growth of the transformants / recombinants.
(ii) Simpler process / less cumbersome, in the presence of chromogenic substrate recombinants are colourless and non recombinants are blue in colour. 
Detailed Answer :
(i) In cloning vector pBR322, ampicillin and tetracycline resistance genes serve as selectable markers. Selectable markers help in the identification and selection of transformed cells from non-transformed cells to distinguish the recombinant cells from the non-recombinant cells.
(ii) The coding sequence of an enzyme β-galactosidase is preferred over antibiotic resistance genes because recombinants can be easily visualised and the process is comparatively
simple and less cumbersome. When the foreign gene is inserted within the β-galactosidase gene, the enzyme β-galactosidase gets inactivated (insertional inactivation). Thus, when the bacteria are grown on a chromogenic substrate, non-recombinants will produce blue-coloured colonies while the recombinants will produce colourless colonies. 

Question. Name and describe the technique that helps in separating the DNA fragments formed by the use of restriction endonuclease.
Answer : Gel electrophoresis.
DNA are negatively charged forced to move towards anode, electric field in agarose gel matrix, separate according to their size / sieving effect, smaller fragments moves faster and farther than the larger. 
Detailed Answer :
Gel electrophoresis, Since DNA fragments are negatively charged molecules, they can be separated by forcing them to move towards the anode under an electric field through a medium / matrix. The most commonly used matrix is agarose which is a natural polymer extracted from sea weeds.
The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves. 

Question. (i) In pBR322, foreign DNA has to be introduced in tet^R region. From the restriction enzymes given below, which one should be used and why :
Pvul, EcoRI, BamHI
(ii) Give reasons, why the other two enzymes cannot be used.
Answer : (i) Bam HI should be used, as restriction site for this enzyme is present in tet^R region.
(ii) Pvu I will not be used as restriction site for this enzyme is present in ampR region (not in tet^R ).
EcoRI will not be used, as restriction site for this enzyme is not present in selectable marker tet^R . 

Question. Mention the role of (i) selectable marker, (ii) Ori and (iii) rop in E. coli cloning vector pBR322.
Answer : (i) Selectable marker : Helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. 
(ii) Ori : Helps to start replication and any piece of DNA when linked to this sequence can be made to replicate within host cell, responsible for controlling the copy number of the linked DNA. 
(iii) Codes for the proteins involved in the replication of the plasmid. 

Question. What is EcoRI ? How does EcoRI differ from an exonuclease ? 
Answer : EcoRI is restriction endonuclease enzyme. 
Exonuclease removes nucleotides from the ends of DNA. 
EcoRI makes cuts at specific position within the DNA. 

Question. (i) Differentiate between exons and introns.
(ii) What is a plasmid ? Why is it selected as a vector ?
Answer : (i) Exons are the coding or expressed sequences that appear in mature or processed RNA, introns are intervening sequences that do not appear in mature or processed RNA / Exons are codons that code for amino acid sequence, introns do not code for amino acids. 
(ii) Autonomously replicating circular DNA / extra chromosomal DNA, exclusively present in bacteria.
Plasmid is selected as vector because it has ability to replicate in the bacterial cell independent of chromosomal DNA and also has high copy number. 

Question. (i) Explain the significance of ‘palindromic nucleotide sequence‘ in the formation of recombinant DNA.
(ii) Write the use of restriction endonuclease in the above process.
Answer : (i) Palindromic nucleotide sequence is the recognition (specific) sequence present both on the vector and on a desired / alien DNA for the action of the same (specific) restriction endonuclease to act upon. 
(ii) Same restriction endonuclease binds to both the vector and the foreign DNA, cut each of the two strands of the double helix at specific points in their sugar phosphate backbone of recognition sequence for restriction endonucleases / palindromic sequence of vector and foreign DNA, to cut strand a little away from the centre of the palindrome sites, creates overhanging stretches / sticky ends. 

Question. Name and explain the technique that helps in the separation of DNA fragments for DNA recombinant technology experiments.
How can these separated DNA fragments be visualised ? 
Answer : Gel electrophoresis, Since DNA fragments are negatively charged, they move towards anode (under an electric field) through a medium / matrix / agarose gel. The fragments separate (resolve) according to their size through sieving effect provided by agarose gel. The separated DNA fragments can be visualised after staining the DNA with ethidium bromide, followed by exposure to UV radiation. 
Detailed Answer :
Electrophoresis is a technique of separation of charged molecules under the influence of an electrical field so that they migrate in the direction of electrode bearing the opposite charge, through a medium / matrix The most commonly used matrix is agarose which is a polysaccharide extracted from sea weeds.
DNA fragments separate according to their size through the pores of agarose gel.
The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (Et + Br) followed by exposure to UV radiation as bright orange colored bands.

Question. Explain the roles of the following with the help of an example each in recombinant DNA technology:
(a) Restriction Enzymes
(b) Plasmids
Answer : (a) It recognizes a specific sequence of base pairs palindromes and cuts the DNA strand at a specific site. 
E.g. EcoRI / Hind II or any other correct example. 
(b) Act as vectors / cloning of desired alien gene / foreign gene. 
E.g. pBR322 / plasmid of Salmonella / plasmid of Agrobacterium / Ti plasmid/ Tumour inducing plasmid. 

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.

Answer : Region to be amplified 

 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.

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. 

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. Unless the vector and source DNA are cut, fragments separated and joined, the desired recombinant vector molecule cannot be created.
(i) How are the desirable DNA sequence cut ?
(ii) Explain the technique used to separate the cut fragments.
(iii) How are the resultant fragments joined to the vector DNA molecule?
Answer : (i) DNA sequences of the vector as well as the source are cut by the same restriction enzyme like EcoRI, in a palindromic sequence.
(The cut ends overhang as sticky ends in the medium.)
(ii) These cut ends fragments are to be extracted from the culture medium using gel electrophoresis.
This has an agarose gel matrix. Fragments are fed in the wells. DNA are negatively charged so, they move towards anode under an electric field through the gel. Smaller fragments move faster, thus separated.
(iii) Fragments are now added to the medium containing the vector DNA.
The sticky ends facilitates the action of the enzyme ligase and join the source DNA to the vector. 

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.