CBSE Class 12 Biology Biotechnology Principles and Processes Assignment Set 01

Question: The controlled use of biological agents, such as live organisms or enzymes from organisms to produce products and processes useful to humans is called as
a) biochemistry
b) molecular biology
c) biotechnology
d) microbiology

Answer: c

Question: In the naming of restriction enzymes, the first letter of the name is derived from ..... A .... and next two letters from the ..... B ..... and fourth letters from the name of ..... C .... of .... D .... from which the enzymes are extracted.
A to D in the statement can be
A B C D
a) genus species strain bacteria
b) species genus strain bacteria
c) genus species variety eukaryote
d) species genus variety eukaryote

Answer: a

Question: EFB stands for
a) European Federation of Biotechnology
b) Eurasian Federation of Biotechnology
c) East Asia Federation of Biotechnology
d) Ethiopian Federation of Biotechnology

Answer: a

Question: Polymerase Chain Reaction (PCR) needs
a) DNA template
b) Primers
c) Taq polymerase
d) All of these

Answer: d

Question: The cutting of DNA by ……… results in the fragments of DNA. Choose the appropriate option.
a) restriction endonucleases
b) exonuclease
c) endonuclease
d) anhydro L-galactose

Answer: a

Question: The specific sequence of DNA that initiate replication of alien DNA in rDNA technology is called as
a) initiation sequence
b) origin of replication
c) origin of DNA
d) initiation of DNA

Answer: b

Question: The enzymes, commonly used in genetic engineering are
a) restriction endonuclease and polymerase
b) endonuclease and ligase
c) restriction endonuclease and ligase
d) ligase and polymerase

Answer: c

Question: Restriction endonuclease binds to DNA and cuts two strands of double helix at specific points in their
a) sugar-phosphate backbone
b) hydrogen bond
c) glycosidic bonds
d) None of the above

Answer: a

Question: Genetic engineering techniques include
a) altering genetic material
b) sequencing genetic material
c) studying genetic material
d) None of the above

Answer: a

Question: Restriction enzyme cuts the DNA strand a little away from the centre of palindrome site between
a) same two bases on same strand
b) same two bases on opposite strand
c) opposite bases on same strand
d) opposite bases on opposite strand

Answer: b

Question: Which of the following methods(s) is used to introduce foreign DNA into plant host cells?
a) Gene gun method
b) Gel electrophoresis
c) Elution
d) Extension

Answer: a

Question: Which of the following option (s) is not correct regarding Eco RI enzyme?
a) Restriction endonuclease enzyme
b) Isolated from Escherichia coli RY13
c) Cuts at specific position within the DNA
d) None of the above

Answer: d

Question: Which of the following techniques is most commonly used to separate DNA molecules by size?
a) Chromatography
b) PCR
c) RFLP
d) Gel electrophoresis

Answer: d

Question: In gel electrophoresis, the separated bands of DNA are cut out and extracted from the gel piece. This step is called
a) elution
b) origin of replication
c) competency
d) transformation

Answer: a

Question: How many fragments will be generated, if a closed circular DNA molecule is digested using a restriction enzyme having six recognition sites on the DNA?
a) 4
b) 6
c) 7
d) 5

Answer: b

Question: In recombinant DNA technique, the term vector refers to a
a) donor DNA, it is identified and picked up through electrophoresis
b) plasmid transfers DNA into host cell
c) collection of entire genome in the form of plasmid
d) enzyme, cuts the DNA at specific sites

Answer: b

Question: Stirred-tank bioreactors are advantageous over shake flasks because they
a) provide high temperature and pH
b) provide better aeration and mixing properties
c) do not allow the entry of CO₂
d) are easy to operate

Answer: b

Question: Which of the following is used in recombinant DNA technique?
a) Cell wall of virus
b) Gene which produces capsid of virus
c) Bacteriophage
d) Capsid of virus

Answer: c

Question: The different basic steps of genetically modifying an organism are given below randomly.
I. Identification of DNA with desirable genes.
II. Transfer of the DNA to its progeny.
III. Maintenance of introduced DNA in the host.
IV. Introduction of identified DNA into the host.
Which of the following represents the correct sequence of steps?
a) I, II, III and IV
b) I, IV, III and II
c) III, IV, II and I 
d) I, III, IV and II

Answer: b

Question: The function of ori in a vector is
a) help in replication of linked DNA
b) control copy number of the linked DNA
c) help in selecting recombinants
d) Both (a) and (b)

Answer: d

Question: If recombinant DNA carrying antibiotic resistance gene (e.g. ampicillin) is transferred into E. coli cell, the host cell is transformed into ampicillin resistant cells. The ampicillin resistant gene in this case is called a
a) vectors
b) plasmid
c) selectable marker
d) cloning sites

Answer: c

Question: A single PCR amplification cycle involves
a) denaturation
b) extension
c) annealing
d) All of these

Answer: d

Question: The method(s) that is/are used to differentiate recombinants and non-recombinants is/are
a) antibiotic affected gene
b) insertional inactivation
c) gene cloning
d) Both (a) and (b)

Answer: b

Question: In insertional inactivation, the recombinant DNA is inserted within the coding sequence of
a) b-galactosidase
b) tetracycline resistant gene
c) restriction enzyme
d) ampicillin resistant gene

Answer: a

Question: The different steps involved in the process of recombinant DNA technology are given below randomly? Arrange these in correct order.
I. Extraction of the desired gene product.
II. Amplification of the gene of interest.
III. Isolation of a desired DNA fragment.
IV. Ligation of the DNA fragment into a vector.
V. Insertion of recombinant DNA into the host.
Correct order is
a) I, II, III, IV and V
b) III, II, IV, V and I
c) II, IV, V, III and I
d) I, IV, V, III and II

Answer: b

Question: Agrobacterium tumefaciens delivers a piece of DNA into dicot plant. The piece of DNA is called as
a) rDNA
b) T-DNA
c) mDNA
d) cDNA

Answer: b

Question: The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called
a) cloning vector
b) vehicle DNA
c) gene carrier
d) All of these

Answer: d

Question: The treatment of host cell with divalent cation leads to the
a) change in permeability of DNA
b) increased efficiency with which DNA enters the bacterium
c) decreased efficiency with which DNA enters the bacterium
d) change in permeability of host

Answer: b

Question: RNA is removed by the treatment with
a) ribonuclease
b) protease
c) chitinase
d) cellulase

Answer: a

Question: Protein encoding gene which is expressed in heterologous host is
a) foreign protein
b) heterologous protein
c) recombinant protein
d) alien protein

Answer: c

Question: Having become an expert on gel electrophoresis, you are asked to examine a gel for a colleague. Where would you find the smallest fragments of DNA?
a) Near the positive electrode, farthest away from the wells
b) Near the negative electrode, close to the wells
c) Near the top, near the negative pole
d) Near the middle they tend to slow-down after the first few minutes

Answer: a

Question: Stirred-tank bioreactors have been designed for the
a) purification of the product
b) addition of preservatives to the product
c) availability of oxygen throughout the biorector
d) ensuring anaerobic conditions in the culture vessel

Answer: c

Question: The components of a bioreactor are
I. an agitator system.
II. an oxygen delivery system.
III. foam control system.
IV. temperature control system.
V. pH control system.
VI. sampling ports to withdraw cultures periodically.
Choose the correct option.
a) I, II, III, IV and V
b) II, IV, V and VI
c) I, II, III, IV and VI
d) All of these

Answer: d

Question. If the bacterium does not have any insert, then the presence of chromogenic substrate, it gives :
(a) Red coloured colonies
(b) Colourless colonies
(c) Blue colonies
(d) Green colonies

Answer : C

Question. Large vessel in which raw materials are biologically converted into specific products, individual enzymes etc using microbial plant, animal or human cell is:
(a) Biotank
(b) Biovessel
(c) Bioreactor
(d) None of the above

Answer : C

Question. The enzymes, which remove nucleotides from the ends of the DNA are :
(a) Exonuclease
(b) Endonuclease
(c) Cellulase
(d) Hydrolase

Answer : A

Question. When a recombinant DNA is inserted within the coding sequence of an enzyme b-galatosidase, it results into inactivation of the enzyme gene this is called :
(a) Insert inactivation
(b) Insertional inactivation
(c) Insertional activation
(d) None of the above

Answer : B

Question. Group of letters that form the same words when read both forward and backward is called :
(a) Palindrome
(b) Same words
(c) Opposite words
(d) None of the above

Answer : A

Question. Which type of ends are produced by EcoRI ?
(a) Blunt ends
(b) Sticky ends
(c) Both (a) and (b)
(d) None of the above

Answer : B

Question. The sequence which is responsible for controlling the copy number of the linked DNA is :
(a) Coding sequence
(b) Promoter sequence
(c) Terminator sequence
(d) Ori

Answer : D

Question. In gel electrophoresis the DNA fragments separate according to size (smaller the fragment size, the faster it moves) this effect is called :
(a) Sieving effect
(b) Movement effect
(c) Size effect
(d) Spooling

Answer : A

Question. Extraction, purification and packaging of products is collectively known as :
(a) Upstream processing
(b) Distillation
(c) Downstream processing
(d) Genetic engineering

Answer : C

Question. The enzymes responsible for restricting the growth of bacteriophage in E-coli were isolated in 1963, these enzyme are :
(a) DNA ligases
(b) Alkaline phosphatases
(c) DNA polymerases
(d) Restriction endonuclease

Answer : D

Question. DNA fragments are
(a) negatively charged
(b) neutral
(c) either positively or negatively charged depending on their size
(d) positively charged.

Answer: A 

Question. A gene whose expression helps to identify transformed cell is known as
(a) vector
(b) plasmid
(c) structural gene
(d) selectable marker.

Answer: D

Question. What is the criterion for DNA fragments movement on agarose gel during gel electrophoresis ?
(a) The smaller the fragment size, the farther it moves.
(b) Positively charged fragments move to farther end.
(c) Negatively charged fragments do not move.
(d) The larger the fragment size, the farther it moves. 

Answer: A

Ques. A foreign DNA and plasmid cut by the same restriction endonuclease can be joined to form a recombinant plasmid using
(a) EcoRI
(b) Taq polymerase
(c) polymerase III
(d) ligase.

Answer: D

Question. Which of the following restriction enzymes produces blunt ends?
(a) SalI
(b) EcoRV
(c) XhoI
(d) HindIII

Answer: B

Question. Which of the following is not a feature of the plasmids?
(a) Transferable
(b) Single-stranded
(c) Independent replication
(d) Circular structure 

Answer: B

Question. Which of the following is a restriction endonuclease?
(a) DNase I
(b) RNase
(c) Hind II
(d) Protease

Answer: C

Question. The introduction of T-DNA into plants involves
(a) exposing the plants to cold for a brief period
(b) allowing the plant roots to stand in water
(c) infection of the plant by Agrobacterium tumefaciens
(d) altering the pH of the soil, then heat-shocking the plants. 

Answer: C

Question. Which vector can clone only a small fragment of DNA?
(a) Bacterial artificial chromosome
(b) Yeast artificial chromosome
(c) Plasmid
(d) Cosmid 

Answer: C

Question. Commonly used vectors for human genome sequencing are
(a) T - DNA
(b) BAC and YAC
(c) expression vectors
(d) T/A cloning vectors.

Answer: B

Question. The colonies of recombinant bacteria appear white in contrast to blue colonies of non-recombinant bacteria because of
(a) insertional inactivation of alpha galactosidase in recombinant bacteria
(b) inactivation of glycosidase enzyme in recombinant bacteria
(c) non-recombinant bacteria containing beta galactosidase
(d) insertional inactivation of alpha galactosidase in non-recombinant bacteria.

Answer: C

Question. DNA fragments generated by the restriction endonucleases in a chemical reaction can be separated by
(a) electrophoresis
(b) restriction mapping
(c) centrifugation
(d) polymerase chain reaction.

Answer: A

Question. A single strand of nucleic acid tagged with a radioactive molecule is called
(a) vector
(b) selectable marker
(c) plasmid
(d) probe. 

Answer: D

Question. For transformation, micro-particles coated with DNA to be bombarded with gene gun are made up of
(a) silver or platinum
(b) platinum or zinc
(c) silicon or platinum
(d) gold or tungsten.

Answer: D

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

Answer: B

Question. In genetic engineering, the antibiotics are used
(a) as selectable markers
(b) to select healthy vectors
(c) as sequences from where replication starts
(d) to keep the cultures free of infection.

Answer: A

Question. Which one of the following represents a palindromic sequence in DNA?
(a) 5′ - GAATTC - 3′ (b) 5′ - CCAATG - 3′
3′ - CTTAAG - 5′ 3′ - GAATCC - 5′
(c) 5′ - CATTAG - 3′ (d) 5′ - GATACC - 3′
3′ - GATAAC - 5′ 3′ - CCTAAG - 5′

Answer: A

Question. Given below is a sample of a portion of DNA strand giving the base sequence on the opposite strands. What is so special shown in it?
5′ _____ GAATTC _____ 3′
3′ _____ CTTAAG _____ 5′
(a) Replication completed
(b) Deletion mutation
(c) Start codon at the 5′ end
(d) Palindromic sequence of base pairs 

Answer: D

Question. There is a restriction endonuclease called EcoRI. What does “co” part in it stand for?
(a) colon
(b) coelom
(c) coenzyme
(d) coli 

Answer: D

Question. Agarose extracted from sea weeds is used in
(a) spectrophotometry
(b) tissue culture
(c) PCR
(d) gel electrophoresis.

Answer: D

Case-based MCQs

I. Read the following text and answer the following questions on the basis of the same :
The term “Biotechnology” refers to the use of living organisms or their products to modify human health and their human environment. For example, ‘testtube’ programme, synthesis of a gene or correcting a defective gene are all part of the biotechnology. The basis of the modern biotechnology are genetic engineering and maintenance of sterile conditions. Genetic engineering is the technique that alter the chemistry of genetic material i.e., DNA and RNA, then this genetic material is introduced into host organisms, which alter the phenotype of the host organism.

Question. The recognition sequence of the first restriction enzyme isolated was ________ base pair long.
(A) four
(B) six
(C) five
(D) two.
Answer : B

Question. The cutting of DNA at specific locations became possible with the discovery of :
(A) Ligases
(B) Restriction enzymes
(C) Probes
(D) Selectable markers.
Answer : B

Question. Discovery of _________ molecule made genetic engineering possible.
(A) Restriction exonuclease
(B) Restriction endonuclease
(C) Ribozyme
(D) DNA polymerase
Answer : B

Question. The specific DNA sequence where EcoRI cuts is :
(A) GATTCG
(B) GAATTC
(C) GTTCAA
(D) TTCCAA.
Answer : B

Question. DNA fragments are :
(A) Positively charged
(B) Negatively charged
(C) Neutral
(D) Either positively or negatively charged depending on their size.
Answer : B

Very Short Answer Questions

Question. Biotechnologists refer to Agrobacterium tumifaciens as a natural genetic engineer of plants.
Give reasons to support the statement. 
Answer. This is because A. tumifaciens can transfer genes naturally by delivering a piece of T-DNA to plant cells. It has a tumour inducing plasmid.

Question. Why is the enzyme cellulase needed for isolating genetic material from plant cells and not from the animal cells? 
Answer. The enzyme cellulase breaks down cellulose which is present in cell walls of plants but absent in animal cells.

Question. Name the compound used for staining the isolated DNA in the gel electrophoresis.
Answer. Ethidium bromide.

Question. What is gene gun?
Answer. The instrument for bombarding micro-projectile particles (gold/tungsten particles) coated with foreign DNA, with great velocity, into a target cell is called gene gun.

Question. How does an alien DNA gain entry into a plant cell by ‘biolistics’ method? 
Answer. In biolistics method, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.

Question. Why EtBr is used in gel electrophoresis inspite of it being highly carcinogenic?
Answer. Ethidium bromide (EtBr) exchanges its visible range of wavelength with the invisible wavelength of DNA, to make it visible under UV light.

Question. Why is it not possible for an alien DNA to become part of a chromosome anywhere along its length and replicate normally? 
Answer. Alien DNA must be linked to ori or origin of replication site to start replication.

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

Question. Write the name of the enzymes that are used for isolation of DNA from bacterial and fungal cells respectively for Recombinant DNA technology.
Answer. Bacterial cell is treated with enzyme lysozyme.
Fungal cell is treated with chitinase.

Question. Which main technique and instrument is used to isolate DNA from any plant cell?
Answer. Centrifugation and centrifuge

Question. By using which vector, the nematode specific genes were introduced into the tobacco host plant. 
Answer. By using Agrobacterium, the nematode specific genes were introduced into the tobacco host plant.

Question. Why do DNA fragments move towards the anode during gel electrophoresis ?
Answer. DNA fragments are negatively-charged. 

Question. Suggest a technique to a researcher who needs to separate fragments of DNA.
Answer. Gel electrophoresis is used to separate DNA fragments.

Question. Mention the role of Restriction Enzymes in Recombinant DNA technology.
Answer. To cut DNA at specific sites / Molecular scissors

Question. Mention the use of gel electrophoresis in biotechnology experiments.
Answer. Cut fragments of DNA can be segregated / separated.

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

Question. Write down the correct order of steps in polymerase chain reaction (PCR).
Answer. A single PCR amplification cycle involves three basic steps : denaturation, annealing and extension.
PCR stands for a polymerase chain reaction in which multiple copies of the DNA segment, or gene of interest is synthesised in vitro.

Short Answer Questions

Question. Explain with the help of an example the relationship between restriction endonuclease and a palindromic nucleotide sequence. 
Answer. Restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.
Restriction endonuclease cuts the strand of DNA a little away from the centre of palindromic nucleotide sequence but between the same two bases on the opposite strands, leaving single stranded portions at the end called sticky ends.

Question. Would you like to choose an exonuclease enzyme 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 the natural source of agarose. Mention one role of agarose in biotechnology.
Answer. The natural source of agarose is sea weed. Agarose is a natural polymer. It is used to develop the matrix for gel electrophoresis. It helps in the separation of DNA fragments based on their size.

Question.  Restriction enzymes present in the cloning site of a vector should not have more than one recognition site. Comment. 
Answer. If the restriction enzymes have more than one recognition site in a vector, then the vector itself will get fragmented on treatment with the restriction enzyme.

Question. Write any four ways used to introduce a desired DNA segment into a bacterial cell in recombinant technology experiments. 
Answer. (i) The desired DNA segment is inserted into a cloning vector and the bacterial cell can be made to take it up after making them competent by treating them with specific concentration of
divalent cations such as calcium.
(ii) Microinjection
(iii) Biolistics
(iv) Disarmed pathogen vector

Question. A plasmid without a selectable marker was chosen as vector for cloning a gene.
Answer. In a gene cloning experiment, first a recombinant DNA molecule is constructed, where the gene of interest is ligated to the vector 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-transformants, because role of selectable marker is in the selection of transformants.

Question. How can DNA segments, separated by gel electrophoresis, be visualised and isolated?
Answer. The separated DNA molecules are visualised only after staining DNA with ethidium bromide followed by exposure to UV radiation. They appear as bright orange coloured bands. The separated bands of DNA (on the gel) are cut from the agarose gel and extracted from the gel piece. This process is called elution.

Question. Why does the ‘insertional inactivation’ method to detect recombinant DNA is preferred to ‘antibiotic resistance’ procedure? 
Answer. In insertional inactivation method, the presence of a chromogenic substrate gives blue coloured
colonies in absence of an insert. Presence of an insert in the enzyme site does not produce colour.
This is because insertional inactivation of the β-galactosidase has taken place due to the insert.
Antibiotic resistance method requires duplicate plating. It is a cumbersome procedure to perform.

Question. (a) A recombinant vector with a gene of interest inserted within the gene of a-galactosidase enzyme, is introduced into a bacterium. Explain the method that would help in selection of recombinant colonies from non-recombinant ones.
(b) Why is this method of selection referred to as “insertional inactivation”?
Answer. (a) Bacteria is grown in a medium with chromogenic substrate, blue coloured colonies show no recombinations and colonies with no blue colour show presence of recombinants.
(b) Gene for the enzyme is inactivated by insertion of foreign 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 to the template, no extension will take place, hence no amplification will occur.

Question. Why and how bacteria can be made ‘competent’? 
Answer. Bacteria are made competent to accept the DNA or plasmid molecules. This is done by treating them with specific concentration of a divalent cation such as calcium to increase pore size in cell wall. The cells are then incubated with recombinant DNA on ice, followed by placing them briefly at 42°C and then putting it back on ice.

Question. DNA being hydrophilic cannot pass through the cell membrane of a host cell. Explain how does recombinant DNA get introduced into the host cell to transform the latter. 
Answer. DNA being a hydrophilic molecule, cannot pass through cell membranes.
- Therefore, the bacteria should be made competent to accept the DNA molecules.
- Competency is the ability of a cell to take up foreign DNA.
- The cell is made competent by the following methods:
(i) Chemical method (ii) Physical method

Question. What modification is done in the Ti plasmid of Agrobacterium tumefaciens to convert it into a cloning vector?
Answer. T-DNA is the only essential part required to make Ti plasmid a cloning vector. The plasmid is disarmed by deleting the tumour inducing genes in the plasmid so that it becomes an effective cloning vector and remove it harmful effect.

Question. What would happen when one grows a recombinant bacterium in a bioreactor forget to add antibiotic to the medium in which the recombinant is growing bacterium?
Answer. In the absence of antibiotic, there will be no pressure on recombinants to retain the plasmid (containing the gene of your interest). Since, maintaining a high copy number of plasmids is a metabolic burden to the microbial cells, it will thus tend to lose the plasmid.

Question. Rearrange the following in the correct sequence to accomplish an important biotechnological reaction:
(a) In vitro synthesis of copies of DNA of interest
(b) Chemically synthesised oligonucleotides
(c) Enzyme DNA-polymerase
(d) Complementary region of DNA
(e) Genomic DNA template
(f) Nucleotides provided
(g) Primers
(h) Thermostable DNA-polymerase (from Thermus aquaticus)
(i) Denaturation of dsDNA
Answer. Correct sequence is
i→ e→ b/g→ g/b→ c/b→ h/c→ f→d→ a 

Question. How can the following be made possible for biotechnology experiments?
(a) Isolation of DNA from bacterial cell.
(b) Reintroduction of the recombinant DNA into a bacterial cell. 
Answer. (a) By treating cell with lysozyme
(b) Microinjection/gene gun

Question. Name the source organism that possesses Taq polymerase. What is so special about the function of this enzyme?
OR
Name the organism from where the thermostable DNA polymerase is isolated. State its role in genetic engineering. 
Answer. Source organism: Thermus aquaticus
The enzyme can tolerate high temperature and is thus thermostable. It does not get denatured during PCR at high temperature.

Question. How are recombinant vectors created? Why is only one type of restriction endonuclease required for creating one recombinant vector? 
Answer. The construction of recombinant DNA is done by linking a gene encoding antibiotic resistance with a native plasmid. These plasmid DNA act as vectors to transfer the piece of DNA attached to it.
Only one type of restriction endonuclease is required for creating recombinant vector because when cut by the same enzyme, the resultant DNA fragments have the same sticky ends, which can be joined together using DNA ligases.

Question. Name the type of bioreactor shown. Write the purpose for which it is used.

Answer. The given bioreactor is the simple stirred tank bioreactor.
Its purpose is large scale production of recombinant protein or enzymes, using microbial plants/ animals/human cells.

Question. (a) Explain how to find whether an E.coli bacterium has transformed or not when a recombinant
DNA bearing ampicillin resistant gene is transferred into it.
(b) What does the ampicillin resistant gene act as in the above case? 
Answer. (a) E.coli bearing transferred recombinant DNA are first grown on ampicillin containing medium and then transferred on to a medium containing tetracycline. The transformants will grow only in ampicillin containing medium and not in tetracycline containing medium. The nontransformants,on the other hand, will grow in both the mediums.
(b) Ampicillin resistant gene acts as a selectable marker and helps in selecting the transformants.

Question. “Cotton bollworms enjoy feeding on cotton plants, but get killed when feed on Bt cotton plant”. Justify the statement.
Answer. Bt-cotton plant is a transgenic plant that produces an insecticide for bollworm. Bacillus thuringiensis is the bacterium that has a Bt gene or Cry gene
which is incorporated in the cotton plant through biotechnological methods and thus, it produces a crystalline product Bt. The product acts as a toxin when ingested by the pests as it gets activated after reaching the alkaline gut of pests and creates pores in the intestine and kills the pests. The gene protects the GMO against budworm, bollworm, beetle, borer, etc.

Question. Explain the role(s) of the following in Biotechnology :
(i) Restriction endonuclease
(ii) Gel - electrophoresis
(iii) Selectable markers in pBR322.
Answer. (i) Cuts at specific position within the DNA / cuts DNA at specific nucleotide / cuts at palindromic nucleotide sequence. 
(ii) Separation of DNA fragments (under the influence of electric field). 
(iii) Helps in identifying and eliminating nontransformants from transformants / selection of transformants. 

Question. When the gene product is required in large amounts, the transformed bacteria with the plasmid inside the bacteria are cultured on a large scale in an industrial fermenter which then synthesizes the desired protein. This product is extracted from the fermenter for commercial use.
(a) Why is the used medium drained out from one side while fresh medium is added from the other? Explain.
(b) List any four optimum conditions for achieving the desired product in a bioreactor.
Answer. (a) To maintain the cells in their physiologically most active log/exponential phase.
(b) Temperature, pH, substrate, salts, vitamins, oxygen.

Question. (i) Why must a cell be made ‘competent’ in biotechnology experiments ? How does calcium ion help in doing so ?
(ii) State the role of ‘biolistic gun’ in biotechnology experiments.
Answer. (i) To take up the (hydrophilic) DNA from the external medium. 
Divalent calcium ions increase the efficiency of the cell to take up foreign DNA through pores in the cell wall.
(ii) To introduce alien DNA into the plant cell by bombarding them with high velocity microparticles (gold or tungsten coated with DNA).

Question. Name the most commonly used bioreactor in biotechnology labs. Mention the most essential components this bioreactor must
have so as to provide the optimum conditions to the culture medium, resulting in production of large volume of desired product.
Answer. Stirred-type Agitator system, O₂ delivery system, foam control system, temperature control system, pH control system.

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 sizes 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. 

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) The 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.

Question. Write the functions of the following in biotechnology.
(i) Polymerase chain reaction technique
(ii) Restriction endonucleases
(iii) Bacterium Thermus aquaticus.
Answer. (i) Multiple copies of gene of interest can be obtained.
(ii) They can cut DNA molecule at a particular point by recognizing a specific sequence of base pairs. Thus they are useful in forming recombinant DNA.
(iii) Thermus aquaticus is the source of Taqpolymerase
which remains active during high temperature induces denaturation of DNA
in PCR technique and therefore allows chain reaction to proceed.

Restriction Enzymes Questions and Answers 

Ques. Which Enzyme Produces sticky Ends in DNA for Foreign DNA Insertion?
Ans. Restriction Endonuclease 

Ques. Name the enzyme that joins the sticky ends to form recombinant DNA.
          5' —— G↓AATTC —— 3'

Ans. DNA Ligase 

Ques. 3' —— CTTAA↑G —— 5'
Name the Enzyme that cuts at the points marked in arrows.
Ans. EcoRI

Ques. Match the following:
a)Ti Plasmid                                 i)Removes wall of Fungus.
b)Biolistics or gene gun                 ii)Beta galactosidase gene
c)Insertional inactivation               iii)insertion of recombinant DNA in Plant cell
d)Chitinase                                   iv)Agarobacterium tumifaciens.

Ans. 
a)Ti Plasmid                                  iv)Agarobacterium tumifaciens
b)Biolistics or gene gun                  iii)insertion of recombinant DNA in Plant cell
c)Insertional inactivation                ii)Beta galactosidase gene
d)Chitinase                                    i)Removes wall of Fungus. 

Ques. Ori sequence is responsible for controlling the copy number of the linked DNA.True/False
Ans. True

Cloning Vectors Questions and Answers 

Ques. Name two selectable markers used in cloning vector.
Ans. Amp-r & tet-r 

Ques. Extention /Denaturation/annealing ,arrange the steps of PCR in proper order according to their occurance.
Ans. Denaturation/annealing/ Extention 

Ques. Exonucleases remove nucleotides from the ends of the DNA.True/False
Ans. True 

Ques. Chomogenic substance produces blue colour by the action of beta galactosidase in non recombinant colony.  True/False
Ans. True 

Ques. Match the followings:-
For the removal of cell wall
a)Lysozyme                i)Plant cells
b)Cellulase                 ii)Fungus
c)Chitinase                 iii)Bacteria

Ans. 
a)Lysozyme                iii)Bacteria
b)Cellulase                  i)Plant cells
c)Chitinase                 ii)Fungus

VSA (1 MARK)

Question 1. A restriction enzyme digests DNA into fragments. Name the technique used to check the progression of this enzyme and separate DNA fragments.
Answer: Gel electrophoresis is the technique used to check the progression of restriction enzyme digestion and separate the resulting DNA fragments based on their size and negative charge.
In simple words: Gel electrophoresis separates DNA fragments by size using electricity.

Exam Tip: Always remember that DNA fragments move towards the anode because DNA is negatively charged.

Question 2. Name two commonly used vectors in genetic engineering.
Answer: Plasmids and bacteriophages are the two most commonly used vectors in genetic engineering.
In simple words: Plasmids and bacteriophages carry foreign genes into host cells.

Exam Tip: Plasmids (like pBR322) are extrachromosomal circular DNA molecules found in bacteria.

Question 3. Some enzymes are considered as molecular scissors in genetic engineering. What is the name assigned to such enzymes?
Answer: Restriction enzymes (specifically restriction endonucleases) are referred to as molecular scissors because they cut DNA at specific recognition sequences.
In simple words: Restriction enzymes act as molecular scissors to cut DNA.

Exam Tip: Distinguish between exonucleases (which remove nucleotides from ends) and endonucleases (which cut at internal specific sites).

Question 4. Write conventional nomenclature of EcoRI.
Answer: In the convention for naming restriction enzymes:
- The first letter comes from the genus name (\(\text{Escherichia}\)).
- The next two letters come from the species name (\(\text{coli}\)).
- The letter 'R' is derived from the name of the strain (RY13).
- The roman numeral 'I' indicates the order in which the enzyme was isolated from that bacterial strain.
In simple words: EcoRI stands for Escherichia coli RY13, isolated first.

Question 5. A linear DNA fragment and a plasmid has three restriction sites for EcoRI how many fragments will be produced from linear DNA and plasmid respectively.
Answer:
- A linear DNA fragment with 3 restriction sites will be cut into 4 fragments.
- A circular plasmid with 3 restriction sites will be cut into 3 fragments.
In simple words: Cutting a straight DNA line 3 times gives 4 pieces, while cutting a circle 3 times gives 3 pieces.

Exam Tip: Number of linear DNA fragments = Number of sites + 1. Number of circular DNA fragments = Number of sites.

Question 6. An extra chromosomal segment of circular DNA of a bacterium is used to carry gene of interest into the host cell. What is the name given to it?
Answer: Plasmid.
In simple words: Plasmids are circular extra-chromosomal DNA used as vectors.

Exam Tip: Plasmids replicate independently of chromosomal DNA.

Question 7. Identify the recognition sites in the given sequences at which E.coli will be cut and make sticky ends.
5'-GAATTC-3'
3'-CTTAAG-5'
Answer: EcoRI cuts between bases G and A on both strands:
5'-G ↓ AATTC-3'
3'-CTTAA ↑ G-5'
In simple words: EcoRI cuts between G and A to create sticky ends.

Exam Tip: EcoRI recognition sequence is 5'-GAATTC-3' which reads the same on both strands when read in 5' to 3' direction (palindromic sequence).

SA-II (2 MARKS)

Question 8. Name two main steps which are collectively referred to as down streaming process. Why is this process significant?
Answer: The two main steps are separation and purification. This process is significant because before a biosynthetic product is marketed as a finished product, it must be subjected to rigorous quality control testing and clinical trials (in the case of drugs) to ensure safety and efficacy.
In simple words: Downstream processing involves separating and purifying the product so it is safe for public use.

Exam Tip: Always mention quality control testing and clinical trials for full marks in this question.

Question 9. How does plasmid differ from chromosomal DNA?
Answer:

In simple words: Plasmids are small circular DNA in bacteria used as vectors, unlike main linear chromosomal DNA.

Exam Tip: Present comparisons in a tabular format for maximum clarity.

Question 10. A bacterial cell is shown in the figure given below. Label the part 'A' and 'B'. Also mention the use of part 'A' in rDNA technology.
Answer:
- Part A: Plasmid
- Part B: Nucleoid (Chromosomal DNA)
Use of part A (Plasmid): Plasmids are used as vectors to transfer the gene of interest into a host cell.
In simple words: A is plasmid and B is nucleoid. Plasmids act as gene transporters.

Exam Tip: Plasmids provide antibiotic resistance genes and serve as vehicles for cloning.

Question 11. Mention two classes of restriction enzymes. Suggest their respective roles.
Answer: The two classes of restriction enzymes are:
1. Exonucleases: They remove nucleotides from the ends of DNA strands.
2. Endonucleases: They cut DNA at specific recognition sites located between the ends of the DNA strand.
In simple words: Exonucleases trim ends, while endonucleases cut inside DNA sequences.

Exam Tip: Genetic engineering predominantly utilizes restriction endonucleases.

Question 12. In the given process of separation and isolation of DNA fragments, some of the steps are missing, Complete the missing steps -
A : Digestion of DNA fragments using restriction endonucleases
↓
B : ..............................................................
↓
C : Staining with ethidium bromide
↓
D : Visualisation in U.V. light
↓
E : .............................................................
↓
F : Purification of DNA fragments.
Answer:
- B: Gel Electrophoresis
- E: Elution (cutting out DNA bands from the agarose gel)
In simple words: Step B is running gel electrophoresis and step E is elution.

Exam Tip: Remember Ethidium bromide gives orange-colored bands under UV light.

SA-I (3 MARKS)

Question 13. Since DNA is a hydrophillic molecule, it cannot pass through cell membranes. Name and explain the technique with which the DNA is forced into (i) a bacterial cell (ii) a plant cell (iii) a animal cell.
Answer:
(i) Bacterial cell: Chemical treatment with divalent cations (like calcium) to increase efficiency, followed by incubation of cells with recombinant DNA on ice, heat shock (pulsing at \(42^\circ\text{C}\)), and placing back on ice.
(ii) Plant cell: Biolistics or gene gun method, where cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA.
(iii) Animal cell: Micro-injection, where recombinant DNA is directly injected into the nucleus of an animal cell.
In simple words: Different cell types need different methods like heat shock, gene gun, or micro-injection to take up DNA.

Exam Tip: Learn all three transformation methods thoroughly as they frequently appear in board exams.

Question 14. How will you obtain purified DNA from a cell?
Answer: To obtain purified DNA:
1. Treat cells with appropriate enzymes to break cell walls: Lysozyme for bacteria, cellulase for plant cells, and chitinase for fungi.
2. Remove cellular macromolecules such as RNA and proteins by treating with ribonuclease and proteases respectively.
3. Precipitate purified DNA by adding chilled ethanol, which results in the separation of fine threads of DNA.
In simple words: Digest cell walls with enzymes, clean out proteins and RNA, and add cold alcohol to precipitate pure DNA threads.

Exam Tip: Note the specific enzyme for each cell type: Lysozyme (bacteria), Cellulase (plants), Chitinase (fungi).

Question 15. In recombinant DNA technology, vectors are used to transfer a gene of interest in the host cells. Mention any three features of vectors that are most suitable for this purpose.
Answer: The three essential features of a suitable cloning vector are:
1. Origin of replication (Ori): A sequence where replication starts and controls the copy number.
2. Selectable marker: A gene (such as antibiotic resistance genes) that helps identify and eliminate non-transformants.
3. Cloning sites: Unique recognition sites for restriction enzymes where foreign DNA can be inserted.
In simple words: A good vector needs an origin of replication, a selectable marker, and unique cloning sites.

Exam Tip: Vector size should also be small so that it is easy to introduce into host cells.

Question 16. Why is “Agrobacterium-mediated genetic engineering transformation” in plants considered as natural genetic engineering?
Answer: Agrobacterium tumefaciens is a pathogen of several dicot plants that naturally delivers a specific piece of DNA known as T-DNA to transform normal plant cells into tumor cells and direct them to produce required chemicals. Genetic engineers have repurposed this natural gene-transfer mechanism by replacing the tumor-causing genes with desired genes of interest without harming the plant.
In simple words: Agrobacterium naturally infects plants and transfers DNA, which scientists use as a tool for genetic engineering.

Exam Tip: Mention T-DNA and the disabling of virulence/tumor genes for complete understanding.

Question 17. Observe the given sequence of nitrogenous bases on a DNA fragment and answer the following question -
5'-CAGAATTCTTA-3'
3'-GTCTTAAGAAT-5'
(a) Name a restriction enzyme which can recognise this DNA sequence.
(b) Write the sequence after digestion.
(c) Why are the ends generated after digestion called sticky ends?
Answer:
(a) EcoRI
(b) After digestion, the sequence splits into:
5'-CAG-3' and 5'-AATTCTTA-3'
3'-GTCTTAA-5' and 3'-GAAT-5'
(c) The ends are called sticky ends because they have single-stranded overhangs that can form hydrogen bonds with complementary cut DNA segments using DNA ligase.
In simple words: EcoRI cuts the sequence, leaving single-stranded sticky overhangs that easily bind with matching DNA.

Exam Tip: Sticky ends facilitate ligation of foreign DNA with vector DNA efficiently.

Question 18. A selectable marker is used in the selection of recombinants on the basis of their ability to produce colour in presence of chromogenic substrate.
(a) Mention the name of mechanism involved.
(b) Which enzyme is involved in production of colour?
(c) How is it advantageous over using antibiotic resistant gene as a selectable marker?
Answer:
(a) Insertional inactivation.
(b) \(\beta\)-galactosidase.
(c) It is advantageous because recombinant colonies can be distinguished from non-recombinant colonies by their color differentiation (blue vs. color-producing/non-producing colonies) in a single plating step, whereas antibiotic resistance selection requires simultaneous plating on two plates containing different antibiotics.
In simple words: Color selection via insertional inactivation is easier because it avoids tedious dual-plate antibiotic testing.

Exam Tip: Recombinants are colorless while non-recombinants give blue color in presence of X-gal.

LA (5 MARKS)

Question 19. The development of bioreactors is required to produce large quantities of products.
(a) Give optimum growth conditions used in bioreactors.
(b) Draw a well labelled diagram of simple stirred - tank bioreactor.
(c) How does a simple stirred - tank bioreactor differ from sparged stirred - tank bioreactor?
Answer:
(a) Optimum growth conditions include optimal temperature, pH, substrate concentration, salts, vitamins, and oxygen levels.
(b) A simple stirred-tank bioreactor typically consists of a cylindrical vessel with a motor-driven agitator (stirrer), flat blade turbine, foam breaker, pH control port, acid/base inlet, temperature control jacket, and sterile air inlet.
(c) In a simple stirred-tank bioreactor, the stirrer facilitates even mixing and oxygen availability throughout the culture. In contrast, a sparged stirred-tank bioreactor has sterile air bubbled through the bottom of the vessel to increase oxygen surface area contact and mixing for larger scale operations.
In simple words: Bioreactors maintain ideal growth conditions; stirred tanks mix culture evenly, while sparged tanks bubble air directly through the liquid.

Exam Tip: Practice drawing the simple stirred-tank bioreactor diagram clearly with labels like agitator, motor, and pH controller.

Question 20. In the given figure, one cycle of polymerase chain reaction (PCR) is shown -
(a) Name the steps A, B and C.
(b) Give the purpose of each of these steps.
(c) State the contribution of bacterium Thermus aquaticus in this process.
Answer:
(a) Step A: Denaturation; Step B: Annealing; Step C: Extension.
(b) Purpose of steps:
- Denaturation: Heating target DNA to high temperature (\(94^\circ\text{C}\)) separates the two complementary strands.
- Annealing: Two oligonucleotide primers hybridize (bind) to each of the single-stranded DNA templates at lower temperature.
- Extension: DNA polymerase extends the primers using provided nucleotides to synthesize new DNA strands.
(c) Thermus aquaticus provides a heat-stable DNA polymerase called Taq polymerase, which remains active at high temperatures required for repeated denaturation cycles without denaturing itself.
In simple words: PCR involves denaturation, annealing, and extension, powered by heat-stable Taq polymerase from Thermus aquaticus.

Exam Tip: Remember the three temperatures for PCR: \(94^\circ\text{C}\) (Denaturation), ~ \(50-60^\circ\text{C}\) (Annealing), and \(72^\circ\text{C}\) (Extension).

Question 21. Study the figure of vector pBR322 given below in which foreign DNA is ligated at the Bam H1 site of tetracycline resistance gene.
(a) Mention the function of rop.
(b) What will be the selectable marker for this recombinant plasmid and why?
(c) Explain transformation.
Answer:
(a) The rop gene codes for proteins involved in the replication of the plasmid.
(b) The selectable marker will be the ampicillin resistance gene (\(\text{amp}^R\)). Since foreign DNA is ligated at the BamHI site located within the tetracycline resistance gene (\(\text{tet}^R\)), the tetracycline gene undergoes insertional inactivation and loses its resistance. Therefore, transformants can be selected using ampicillin resistance.
(c) Transformation is the phenomenon by which DNA isolated from one organism is introduced into another recipient cell, allowing the recipient cell to acquire new properties or traits of the donor organism.
In simple words: Rop helps plasmid replication, ampicillin acts as the functional marker due to inactivated tetracycline resistance, and transformation is taking up foreign DNA.

Exam Tip: Understand insertional inactivation clearly: the gene where foreign DNA is inserted becomes non-functional.