1. Which one of the following techniques made it possible to genetically engineer living organisms?
(a) Recombinant DNA techniques
(b) X-ray diffraction
(c) Heavier isotope labelling
(d) Hybridisation
Answer (a)
2. In genetic fingerprinting, the ‘probe’ refers
to ––––––
(a) a radioactively labelled single stranded DNA molecule.
(b) a radioactively labelled single stranded RNA molecule.
(c) a radioactively labelled double stranded RNA molecule.
(d) a radioactively labelled double stranded DNA molecule.
Answer (a) : For DNA fingerprinting special single stranded DNA-probes are made in the laboratory. DNA-probes contain repeated sequences of bases complementary to those on VNTRs. These probes are made radioactive by labelling with radioactive isotopes. This step helps in detecting DNA fingerprints or variable number of tandem repeats (VNTRs).
3. Following statements describe the characteristics of the enzyme restriction endonuclease. Identify the incorrect statement.
(a) The enzyme recognises a specific palindromic nucleotide sequence in the DNA.
(b) The enzyme cuts DNA molecule at identified position within the DNA.
(c) The enzyme binds DNA at specific sites and cuts only one of the two strands.
(d) The enzyme cuts the sugar-phosphate backbone at specific sites on each strand.
Answer (c) : The restriction endonuclease enzyme inspects the length of a DNA sequence. Once it recognises specific sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar phosphate backbone. Special sequence in the DNA recognised by restriction endonuclease is called palindromic nucleotide sequence.
4. 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 a
5. Given below are four statements pertaining to separation of DNA fragments using gel electrophoresis. Identify the incorrect statements.
(i) DNA is negatively charged molecule and so it is loaded on gel towards the anode terminal.
(ii) DNA fragments travel along the surface of the gel whose concentration does not affect movement of DNA.
(iii) Smaller the size of DNA fragment larger is the distance it travels through it.
(iv) Pure DNA can be visualized directly by exposing UV radiation.
Choose the answer from the options given below.
(a) (i), (iii) and (iv) (b) (i), (ii) and (iii)
(c) (ii), (iii) and (iv) (d) (i), (ii) and (iv)
Answer (d)
6. The DNA fragments separated on an agarose gel can be visualised after staining with
(a) acetocarmine (b) aniline blue (c) ethidium bromide (d) bromophenol blue.
Answer (c) The separated DNA fragments can be seen only after staining them with a compound known as ethidium bromide (EtBr) followed by exposure to UV radiation as bright orange coloured bands.
7. 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) : Electrophoresis is a technique used for the separation of substances of different ionic properties. Since the DNA fragments are negatively charged molecules, they can be separated by allowing them to move towards the anode. DNA fragments move towards the anode according to their molecule size through the pores of agarose gel. Thus, the smaller fragments move farther away as compared to larger fragments.
8. Which of the following restriction enzymes produces blunt ends?
(a) SalI
(b) EcoRV
(c) XhoI
(d) HindIII
Answer (b) : EcoRV is a type II restriction endonuclease isolated from certain strains of E.coli. It creates blunt ends. It recognises the palindromic sequence of 6 bases as shown here:
9. Which of the following is a restriction endonuclease?
(a) DNaseI (b) RNase (c) HindII (d) Protease
Answer (c) : HindII is the first restriction endonuclease. It was isolated from Haemophilus influenzae Rd. It always cut DNA at specific position producing blunt ends. DNase I is an endonuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide non-specially. RNase is a type of nuclease that catalyses the degradation of RNA into smaller components. It can be endoribonuclease or exoribonuclease. A protease is an enzyme that perform proteolysis, i.e., protein catabolism by hydrolysis of the peptide bonds.
10. Which of the following is not a feature of the plasmids?
(a) Transferable
(b) Single-stranded
(c) Independent replication
(d) Circular structure
Answer (b) : Plasmids are extra-chromosomal, self-replicating, usually circular, double-stranded DNA molecules that serve as vectors which carry foreign DNA segment and replicate inside host cell.
11. Most suitable method of introducing alien DNA into a plant cell is
(a) lipofection (b) biolistics
(c) heat shock method (d) microinjection.
Answer (b) : Biolistics technique also known as ballistic method of DNA delivery has general applicability to plant species and can be used to deliver DNA into virtually all tissues.
12. The DNA molecule to which the gene of interest is integrated for cloning is called
(a) template (b) carrier (c) transformer (d) vector.
Answer (d) : Vector is a DNA molecule that carries a foreign DNA segment and replicates inside a host cell. The vector DNA and foreign DNA carrying gene of interest are cut by the same restriction endonuclease enzyme to produce complementary sticky ends. With the help of DNA ligase enzyme, the complementary sticky ends of the two DNAs are joined to produce a recombinant DNA (rDNA), which is then introduced into the host cell.
13. Restriction endonucleases are
(a) used for in vitro DNA synthesis
(b) synthesised by bacteria as part of their defence mechanism
(c) present in mammalian cells for degradation of DNA when the cell dies
(d) used in genetic engineering for ligating two DNA molecules.
Answer (b)
15. Select the wrong statement.
(a) The presence of chromogenic substrate gives blue colour colonies, if the plasmid in the bacteria does not have an insert.
(b) Retroviruses in animals have the ability to transform normal cells into cancerous cells.
(c) In microinjection, cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA.
(d) Since DNA is a hydrophilic molecule it cannot pass through cell membranes.
Answer (c) : In microinjection method of DNA transfer, foreign DNA is directly injected into the nucleus of animal cell or plant cell by using micro needles or micro pipettes. It is used in oocytes, eggs and embryo. In gene gun method tungsten or gold particles coated with foreign DNA are bombarded into target cell at very high velocity.
16. Which vector can clone only a small fragment of DNA?
(a) Bacterial artificial chromosome
(b) Yeast artificial chromosome
(c) Plasmid
(d) Cosmid
Answer (c)
17. Identify the DNA segment which is not a palindromic sequence.
(a) 5′ GGATCC 3′ (b) 5′ GAATTC 3′
3′ GGTACC 5′ 3′ CTTAAG 5′
(c) 5′ GCGGCCGC 3′ (d) 5′ CCCGGG 3′
3′ CGCCGGCG 5′ 3′ GGGCCC 5′
Answer (a) : Palindromic nucleotide sequence in the DNA molecule is the sequence of base pairs that reads same when orientation of reading is same, i.e., 5′ to 3′ on both strands or 3′ to 5′ on both strands. Hence, (a) is the correct answer, since it does not read same from both the sides.
18. The colonies of recombinant bacteria appear white in contrast to blue colonies of nonrecombinant 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) : The presence of restriction sites within the markers tetr and ampr of plasmid permits an easy selection for cells transformed with recombinant plasmid. Insertion of the DNA fragment into the plasmid makes antibiotic resistance genes nonfunctional, for example, insertion of the DNA fragment into the plasmid (pBR322) using Pst I or Pvu I makes ampr nonfunctional. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline. This process, however, becomes burdensome because it requires simultaneous plating on two plates having different antibiotics. Thus, alternative selectable marker is developed to differentiate recombinants and non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substance. Here, a recombinant DNA is inserted in the coding sequence of an enzyme b-galactosidase. pUC18 plasmid contains this gene which allows it to produce b-galactosidase which degrades certain sugars and produces a blue pigment when exposed to specific substrate analog. If the plasmid in the bacterium does not have an insert, i.e., is non-recombinant, the presence of chromogenic substrate gives blue coloured colonies. Presence of insert in the plasmid in recombinant bacterium does not produce any colour, such bacterial colonies are marked as recombinant colonies.
20. 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) : A gene or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal such as gold or tungsten coated with plasmid DNA. The device is used to transform almost any type of cell including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.
21. The enzymes which are absolutely necessary for recombinant DNA technology are
(a) restriction endonucleases and topoisomerases
(b) endonucleases and polymerases
(c) restriction endonucleases and ligases
(d) peptidases and ligases.
22. 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)
23. 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) : The enzyme restriction endonuclease EcoRI is found in the colon bacteria E. coli. So, here ‘co’ stands for coli. According to nomenclature of restriction enzyme, the first letter used for the enzyme is the first letter of the genus name (in italics) of the bacterium, then comes the first two letters of its species (also in italics), next is the strain of the organism. At last is a Roman numeral signifying the order of discovery. Here, the enzyme EcoRI was isolated from the bacterium Escherichia coli (co), strain RY13(R) and it was first endonuclease (I) isolated from E.coli.
24. Agarose extracted from sea weeds is used in
(a) spectrophotometry (b) tissue culture
(c) PCR (d) gel electrophoresis.
Answer (d)
25. In genetic engineering, a DNA segment (gene) of interest, is transferred to the host cell through a vector. Consider the following four agents (i-iv) in this regard and select the correct option about which one or more of these can be used as a vector/vectors.
(i) Bacterium (ii) Plasmid
(iii) Plasmodium (iv) Bacteriophage
(a) (i), (ii) & (iv) (b) (i) only
(c) (i) & (iii) (d) (ii) & (iv)
Answer (d) : Plasmid and bacteriophage are used as vectors in genetic engineering. Plasmid is an autonomously replicating circular extra chromosomal DNA found in bacteria. They can be transferred from cell to cell in a bacterial colony. This characteristic is being used in biotechnology for transferring desirable gene into target gene of the host. Whereas, bacteriophage is a bacterial virus which can infect it, quickly multiply within and destroy their host (virus) cells. During infection bacteriophages inject their DNA into these cells. The injected DNA selectively replicate and are expressed in the host that results in a multiplication of phages that ultimately burst out of the cell (by lysis). This ability of transferring DNA from the phage genome to specific host during infection process gave scientists the idea that specially designed phage vectors could be used for gene cloning.
26. Which one of the following is used as vector for cloning genes into higher organisms?
(a) Baculovirus
(b) Salmonella typhimurium
(c) Rhizopus nigricans
(d) Retrovirus
Answer (d) : Retroviruses in animals have the ability to transform normal cells into cancerous cells. We have transformed these pathogens into useful vectors for delivering genes of interest to humans. Retroviruses have been disarmed and are now used to deliver desirable genes into animal cells. So, once a gene or a DNA fragment has been ligated into a suitable retroviral vector it is transferred into a bacterial, plant or animal host (where it multiplies).
27. DNA precipitation out of a mixture of biomolecules can be achieved by treatment with
(a) chilled chloroform
(b) isopropanol
(c) chilled ethanol
(d) methanol at room temperature.
Answer (c) : In order to cu t the DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules. Since the DNA is enclosed by the membranes, we have to break the cell open to release DNA and other macromolecules like RNA, proteins, polysaccharides and lipids. It is obtained by treating the bacterial cells/plant or animal tissue with enzymes. Other molecules are removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol.
28. Which one of the following equipments is essentially required for growing microbes on a large scale, for industrial production of enzymes?
(a) Bioreactor
(b) BOD incubator
(c) Sludge digester
(d) Industrial oven
Answer (a)
29. The process of separation and purification of expressed protein before marketing is called
(a) downstream processing
(b) bioprocessing
(c) postproduction processing
(d) upstream processing.
Answer (a) : After the formation of the product in the bioreactor, it undergoes some processes before a finished product is ready for marketing. The process includes separation and purification of products which are collectively called downstream processing. The product obtained is subjected to quality control, testing and kept in suitable preservatives.
30. Stirred-tank bioreactors have been designed for
(a) purification of product
(b) addition of preservatives to the product
(c) availability of oxygen throughout the process
(d) ensuring anaerobic conditions in the culture vessel.
Answer (c) : 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.
31. Match the items in column I with their uses in column II and choose the right option.
Column I Column II
A. ELISA (i) Antigen-antibody interaction
B. PCR (ii) Gene amplification
C. Biolistics (iii) Direct introduction of recombinant DNA
(a) A - (r), B - (s), C - (q), D - (p)
(b) A - (r), B - (q), C - (s), D - (p)
(c) A - (q), B - (r), C - (s), D - (p)
(d) A - (q), B - (s), C - (r), D - (p)
Answer (a)
Case based MCQs
Case I : Read the following passage and answer any four questions from 41 to 45 given below:
Rama lives in a society where a robbery occurred last night. Robbers came into the flat and murdered the old lady residing there. Police came and restricted the entry into the flat. They took samples from the room, where the dead body was found. While examining, they found that there is some blood and tissue in the nails of old lady. According to their observation, police filtered out their inspection to three suspects viz. servant, cook and milkman. Finally after two days of robbery, police caught the criminal. It was the old lady’s cook. Rama was amazed to see that how quickly police completed and shut the case. She asked the inspector that how they did it? The police man told her that it become possible due to the sample collected from the victim, that lead them to the criminal. The sample taken from nail scraping was amplified using PCR and then tested.
41. What technique was used by the police to identify the criminal?
(a) DNA fingerprinting (b) Gel electrophoresis
(c) Molecular diagnosis (d) Cloning
Answer (a) : DNA fingerprinting is one of highly accurate application of biotechnology. It is helpful in solving crime, legal disputes, establishing identity of criminal or parents, etc.
42. In PCR, the temperature used to denature the DNA is about
(a) 76°C (b) 25°C (c) 95°C (d) 40°C.
Answer (c) In PCR, during denaturation, the target DNA is heated at high temperature resulting in the separation of the two strands.
43. Which of the following statements regarding PCR is correct?
(a) Taq polymerase, which is isolated from bacterium Thermus aquaticus is stable at low temperature only.
(b) With the help of DNA ligase, the complementary sticky ends of the DNA are joined to produce a rDNA.
(c) Since the sequence of primers are complementary to 5′ end of the template DNA, they anneal to it.
(d) DNA purified from the cell is precipitated by adding hot ethanol.
Answer (b) : Taq polymerase isolated from bacterium Thermus aquaticus is stable at high temperature. Sequence of primers are complementary to 3′ end of the template. Purified DNA is precipitated by adding chilled ethanol.
44. Taq polymerase synthesises DNA region between the primers using
(a) Mg2+ (b) dNTPs
(c) DNA ligase (d) both (a) and (b).
Answer (d)
45. Given below are steps of polymerase chain reaction.
Select the option that correctly mention the sequence in which they occur.
(a) (ii) → (iii) → (i) (b) (i) → (ii) → (iii)
(c) (iii) → (i) → (ii) (d) (ii) → (i) → (iii)
Answer (a) : Three steps of PCR are : denaturation, annealing and extension.
Case II : Read the following passage and answer any four questions from 46 to 50 given below:
46. Reverse transcriptase enzyme was discovered by
(a) Temin and Baltimore
(b) Cohen and Boyer
(c) Arber and Nathan
(d) Paul Berg.
Answer (a)
47. During cDNA formation, what would happen if DNA formed by reverse transcriptase is not treated with the alkali?
(a) cDNA will not be digested
(b) mRNA will not be digested
(c) Hydrogen bonds will not form between base pairs
(d) mRNA will not be formed.
Answer (b) : The cDNA formation involves the alkaline denaturation of the mRNA-cDNA hybrid. The double stranded DNA molecule formed after the activity of reverse transcriptase is treated with alkali to digest mRNA.
48. Enzyme that helps in the formation of double stranded cDNA is
(a) DNA synthetase (b) ligase (c) DNA polymerase (d) helicase.
Answer (c) : A cDNA strand is formed on the separated single stranded DNA template with the help of DNA polymerase enzyme.
49. DNA polymerase can be obtained form
(a) retrovirus (b) Agrobacterium
(c) tobacco mosaic virus (d) Thermus aquaticus.
Answer (d)
1. Who is considered as the “father of genetic engineering”?
Answer:- Genetic engineering was started by Paul Berg (1972) when he was able to introduce a gene of SV-40 into a bacterium. He is often considered as “father of genetic engineering”.
2. Name the host cells in which microinjection technique is used to introduce an alien DNA.
Answer:- Microinjection technique is used to introduce an alien DNA directly into the nucleus of the animal host cells such as oocytes, eggs and embryo.
3. What is the role of sterile air bubbles in the sparged stirred-tank bioreactor?
Answer:- The sterile air bubbles in the sparged stirred-tank bioreactor increases the surface area for oxygen transfer.
4. Mention the source of thermostable DNA polymerase.
Answer:- Thermophilic bacterium Thermus aquaticus is the source of thermostable DNA Taq polymerase.
5. Name the material used as matrix in gelelectrophoresis and mention its role.
Answer: Most commonly used matrix in DNA gel electrophoresis is agarose. It provides sieving effect for separation of DNA fragments according to their size.
6. What are synthesising enzymes?
Answer: The enzymes that are used to synthesise DNA strands on suitable templates are called synthesising enzymes. They are of two types : DNA polymerase and reverse transcriptase.
7. State what happens when an alien gene is ligated at SalI site of pBR322 plasmid.
Answer: When an alien gene is ligated at SalI site of pBR322, the gene tetR becomes non functional and plasmid loses its tetracycline resistance. Hence, the cell possessing such recombinant pBR322 will not be able to grow on tetracycline.
8. What are the drawbacks of stirred tank bioreactors?
Answer: The main drawback of stirred-tank bioreactor is that it is relatively expensive to run, which is mainly due to high energy requirements.
9. Why do DNA fragments move towards the anode during gel-electrophoresis?
Answer: DNA is a negatively charged molecule hence during gel electrophoresis it moves towards anode (positive electrode) under the influence of electrical field.
10. During the isolation of DNA, how can you remove RNA and protein from the DNA solution?
Answer:- RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease.
11. Why is ‘plasmid’ an important tool in biotechnology experiments?
Answer: Plasmid have the ability to replicate within bacterial cells independent of the control of chromosomal DNA and have high copy number, therefore any alien DNA ligated to it, also multiplies to equal the copy number of plasmids. So, it is used as a vector in gene cloning experiments and thus, plays a role of an important tool in biotechnology.
12. Write the role of ‘ori’ and ‘restriction’ site in a cloning vector pBR322.
Answer: Origin of replication (ori) site in cloning vector pBR322 is a sequence from where replication starts. Any piece of DNA when linked to this sequence can be made to replicate within host cell. Restriction site within the markers tetR and ampR genes permit an easy selection for cells transformed or the recombinant pBR322.
13. Name the natural source of agarose. Mention one role of agarose in biotechnology.
Answer: Agarose is commonly used as matrix in agarose gel electrophoresis. It is extracted from sea weeds. In recombinant DNA technology, agarose is used to separate DNA fragments according to their sizes.
14. What are the two main discoveries that led to the built of genetic engineering ?
Answer: Genetic engineering is the technique to form recombinant DNA and then to introduce recombinant DNA into appropriate host. Two main discoveries which led to genetic engineering are :
(i) Discovery of plasmid by William Hays and Joshua Lederberg in 1952.
(ii) Discovery of restriction endonuclease by Arber in 1962.
15. A recombinant DNA is formed when sticky ends of vector DNA and foreign DNA join. Explain how the sticky ends are formed and get joined.
Answer: When restriction enzymes cut the strand of DNA a little away from the centre of the palindromic sites, between the same two bases on the opposite strands, it leaves single stranded portions at the ends. This forms overhanging stretches called sticky ends on each strand. They are called sticky as they form hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase.
16. Why is it essential to maintain sterile condition in biotechnological processes?
Answer:- Sterile condition enable growth of only the desired microbe/ eukaryotic cell in large quantities for the biotechnological products like antibiotics, enzymes, etc.
17. Explain any two methods of vectorless gene transfer.
Answer: Vectorless gene transfer is a method to introduce recombinant DNA into recipient cells of host without involving carrier molecule.
Two methods of vectorless gene transfer are: (i) Microinjection : It is the introduction of foreign gene into plant cell or animal cell by using microneedles or micropipettes.
(ii) Electroporation : In this method, electrical impulses induce transient pores in the plant cell membrane through which the DNA molecules are incorporated into the plant cells.
18. State the role of UV-light and ethidium bromide during gel electrophoresis of DNA fragments.
Answer: DNA fragments can be seen only after staining. Ethidium bromide is used to stain DNA fragments followed by exposure to UV radiation. This gives bright orange colour to DNA fragments which helps to view separated DNA fragments.
19. Rearrange the following in the correct sequence to accomplish an important biotechnological reaction :
(i) Denaturation of ds-DNA
(ii) Chemically synthesised oligonucleotides
(iii) Primers
(iv) Complementary region of DNA
(v) Thermostable DNA polymerase (from Thermus aquaticus)
(vi) Nucleotides provided
(vii) Genomic DNA template
(viii) In vitro synthesis of copies of DNA of interest
(ix) Enzyme DNA-polymerase.
Answer The correct sequence to accomplish biotechnological reaction is :
Denaturation of ds DNA
↓
Genomic DNA template
↓
Chemically synthesised oligonucleotides (Primers)
↓
Complementary regions of DNA
↓
Thermostable DNA polymerase (Enzyme polymerase) (from Thermus aquaticus)
↓
dNTPs (Nucleotides) provided
↓
In vitro synthesis of copies of DNA of interest
20. Why are genes encoding resistance to antibiotics considered useful selectable markers for E. coli cloning vector? Explain with the help of one example.
Answer: Genes encoding resistance to antibiotics are considered useful selectable markers for E. coli cloning vector because they help in selecting transformant cell from non-transformant ones.
The genes encoding resistance to antibiotics such as tetracycline, ampicillin, kanamycin or chloramphenicol, etc. are useful selectable markers for E. coli. The common E. coli cells are not resistant to any of these antibiotics. Plasmid pBR322 has two antibiotic resistance genes – ampicillin resistance (ampR) and tetracycline resistance (tetR) which are considered useful for selectable markers. The presence of restriction sites within the markers tetR and ampR permits an easy selection for cells transformed with the recombinant pBR322. For example, insertion of the DNA fragment into the plasmid using enzyme PstI or PvuI places the DNA insert within the gene ampR. This makes ampR nonfunctional. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline.
21. Prepare a flow chart in formation of recombinant DNA by the action of restriction endonuclease enzyme EcoRI.
Answer
22. Refer to the given figure and answer the following questions.
(a) Identify the labelled part A and mention its function.
(b) What does B and C represent?
(c) Briefly describe the process shown in the given figure.
Answer:- (a) Labelled part A represents the enzyme reverse transcriptase. Reverse transcriptase is used to synthesise DNA or complementary DNA by using mRNA as template.
(b) The labelled part B represents the separated singlestranded complementary DNA after the RNA-DNA complex is treated with alkaline phosphatase enzyme. The labelled part C is the newly formed double-stranded cDNA. (c) This diagram represents the synthesis of complementary DNA or cDNA from RNA. With the help of the enzyme reverse transcriptase, an RNA-DNA complex is formed from RNA (usually mRNA) where RNA acts as the template. The mRNA in the RNA-DNA complex is digested in the presence of alkaline phosphatase enzyme. A cDNA is formed on the separated single-stranded DNA template with the help of the enzyme DNA polymerase.
23. Differentiate between rDNA and cDNA.
Answer Differences between rDNA and cDNA are as follows:
24. How does b-galactosidase coding sequence act as a selectable marker? Why is it a preferred selectable marker to antibiotic resistance genes? Explain.
Answer: Some genes called selectable markers help in selecting those host cells which contain the vectors and eliminating the non-transformants. b-galactosidase is an alternative selectable marker developed to differentiate recombinants and non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substance. A recombinant DNA is inserted in the coding sequence of an enzyme b-galactosidase. This causes inactivation of the enzyme which is called insertional inactivation. If the plasmid in the bacterium does not have an insert, the presence of a chromogenic substrate gives blue coloured colonies.
25. What are bioreactors ? List five growth conditions that a bioreactor provides for obtaining the desired product.
Answer: A bioreactor is a device in which raw materials are biologically converted into specific products by microbes, plant/animal cells etc. These are used for food processing, fermentation, waste treatment, etc.
26. How and why is the bacterium Thermus aquaticus employed in recombinant DNA technology ? Explain.
Answer:Taq DNA polymerase isolated from thermophilic bacterium Thermus aquaticus synthesises the DNA region from gene of interest between the primers, using dNTPs (deoxynucleotide triphosphates) and Mg2+. The primers are extended towards each other so that the DNA segment lying between the two primers is copied. It is stable even at high temperatures.
27. Give reasons why:
(a) DNA cannot pass into a host cell through the cell membrane.
(b) Proteases are added during isolation of DNA for genetic engineering.
(c) Single cloning site is preferred in a vector.
Answer: (a) DNA is a hydrophilic molecule, so it cannot pass into a host cell through cell membrane. The cell membrane consists of lipid bilayers that are generally impermeable to hydrophilic molecules.
28. (a) Why must a cell be made ‘competent’ in biotechnology experiments? How does calcium ion help in doing so?
(b) State the role of ‘biolistic gun’ in biotechnology experiments.
Answer: (a) Competent host is essential for transformation with recombinant DNA. Since DNA is a hydrophilic molecule, it cannot pass through membranes, so the bacterial cells must be made capable to take up DNA i.e., made competent. This is done by treating them with a specific concentration of a divalent cations, such as calcium which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA (rDNA) can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
29. Name the technique to obtain multiple copies, of a DNA segment of interest, synthesized in vitro. Name two sets of primers that are necessary for reaction to occur. Mention three diagnostic applications of this technique.
Answer: PCR (Polymerase Chain Reaction).
30. Explain the importance of
(i) ori, (ii) ampR and (iii) rop in the E.coli vector shown below.
Answer: (i) ori : ori is the origin of replication. This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within host cells. It controls the copy number of the linked DNA.
31. Explain the role of Ti plasmids in biotechnology.
Answer: Agrobacterium tumefaciens is a soil-inhabiting bacterium that may invade growing plants at the junction of root and stem, where it can cause a cancerous growth known as a crown gall. A. tumefaciens contains Ti plasmid which carries gene for tumour formation for using Agrobacterium tumefaciens as a cloning vector researchers deleted the genes which governs auxin and cytokinin production (the oncogene) from T-DNA of Ti plasmid, it is known as disarming. After disarming, this T-DNA is inserted into chromosomes of the host plant where it produces copies of itself.
Restriction nucleases act as molecular scissors or chemical scalpels. Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds
its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones. Each restriction
endonuclease recognises a specific palindromic nucleotide sequence in the DNA.
The capital letter E comes from the genus Escherichia. The letters co are derived from the species name coli. The letter R is from RYI3 (strain). The Roman number I indicates that it
was the first enzyme isolated from the bacterium E. coli RYI3.
(b) EcoRI is a restriction endonuclease enzyme it recognises
base sequences
5′-GAATTC-3′
3′-CTTAAG-5′ in DNA duplex.
(c) It cut each of the two strands between G and A producing sticky ends.
Answer: (i) The palindromic sequence in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
(ii) The palindrome sequence in the given DNA strand is :GAATTC
CTTAAG
This is the recognition sequence for restriction enzyme EcoRI.
(iii) Each restriction endonucleases recognise a specific palindromic nucleotide sequences in the DNA. Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. These are overhanging stretches called sticky ends on each strand. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.
Long Answer Type Questions (LA)
36. What is cloning vector ? Why is it used ? Explain the technique of using such a vector in E.coli.
Answer: The cloning vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell. These are plasmids, cosmids, phagemids, yeast artificial
chromosome (YAC), bacterial artificial chromosome (BAC), transposons and virus.
Cloning vector carry rDNA and they generally have high copy number, they can produce multiple number of required gene. Vectors help in easy linking of foreign DNA and in selection of recombinants from non recombinants. The entire procedure of gene cloning or recombinant DNA technology may be classified into the following six steps
for the convenience in description and on the basis of the chief activity performed.
(i) Production and isolation of the DNA fragments to be cloned.
(ii) Insertion of the isolated gene in a suitable vector to obtain recombinant DNA. Introduction of the recombinant DNA into a suitable organism/cell (usually E.coli) called host (transformation).
(iv) Selection of the transformed host cells, and identification of the clone containing the desired gene/DNA fragment.
(v) Multiplication/expression of the introduced gene in the host.
37. If a desired gene is identified in an organism for some experiments, explain the process of the following :
(a) Cutting of desired gene at specific locations.
(b) Synthesis of multiple copies of the desired gene.
Answer: (a) Desirable DNA sequences are cut by the use of enzyme restriction endonuclease. The restriction enzymes cut the strand of DNA a little away from the centre of the
palindromic sites, between the same two bases on the opposite strands, it leaves single stranded portions at the ends. This forms overhanging stretches called sticky ends oneach strand. They are called sticky as they form hydrogen bonds with their complementary cut counterparts. The stickiness of the ends facilitates the action of the enzyme DNA ligase.
(b) The three steps involved in each cycle of PCR are :
(i) Denaturation
(ii) Annealing
(iii) Extension
PCR is based on the principle that a DNA molecule, when subjected to high temperature, splits into two strands due to denaturation. These single stranded DNA molecules are
then converted to original double stranded molecules, in the presence of enzyme DNA polymerase. A double stranded molecule of DNA is duplicated in this way and multiple copies of original DNA sequence can be generated by repeating the process several times. Such repeated amplification is achieved by the use of thermostable DNA polymerase (isolated from Thermus aquaticus), which remain active during the high
temperature induced denaturation of double stranded DNA.
38. (a) Mention the role of vectors in recombinant DNA technology. Given any two examples.
(b) With the help of diagrammatic representation only, show the steps of recombinant DNA technology.
Answer: The cloning vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell. These are plasmids, cosmids, phagemids, yeast artificial
chromosome (YAC), bacterial artificial chromosome (BAC), transposons and virus.
Cloning vector carry rDNA and they generally have high copy number, they can produce multiple number of required gene. Vectors help in easy linking of foreign DNA and in selection of recombinants from non recombinants. The entire procedure of gene cloning or recombinant DNA technology may be classified into the following six steps
for the convenience in description and on the basis of the chief activity performed.
(i) Production and isolation of the DNA fragments to be cloned.
(ii) Insertion of the isolated gene in a suitable vector to obtain recombinant DNA. Introduction of the recombinant DNA into a suitable organism/cell (usually E.coli) called host (transformation).
(iv) Selection of the transformed host cells, and identification of the clone containing the desired gene/DNA fragment.
(v) Multiplication/expression of the introduced gene in the host.
(b) Diagram showing various steps of recombinant DNA technology is given below:
