CHAPTER 11.

BIOTECHNOLOGY-PRINCIPLES AND PROCESSES

Q. 1. Can you list 10 recombinant proteins which are used in medical practice? Find out where
they are used as therapeutics.
Ans: Ten such recombinant proteins with their medical use are as follows:

Q. 2.* State what happens when an alien gene is ligated at the Sal I site of pBR322 plasmid?
Ans: When an alien gene is ligated at the Sal I site of tetracycline resistance gene in the vector
pBR322
plasmid, the recombinant loses tetracycline resistance because of insertion of a foreign DNA.
Q. 3.* State what happens when an alien gene is ligated at Pvu I site of pBR322 plasmid?
Ans: When an alien gene is ligated at the given site, the recombinant plasmid loses ampicillin
resistance.
Q. 4. Do eukaryotic cells have restriction endonuclease? Justify your answer.
Ans: Certainly not. The eukaryotic cells do not have restriction endonuclease. This is because the
DNA of eukaryotes is highly methylated by a modification enzyme, called methylase.
Methylation protects the DNA from the activity of restriction enzymes. These enzymes are
present in prokaryotic cells where they help to prevent the invasion of DNA by virus.
Q. 5.* Explain with the help of suitable example, the naming of a restriction endonuclease.
Ans: Considering the example of EcoRI, the Eco stands for the genus and the species of the
prokaryotic cell from which the enzyme has been isolated that is E.coli, R stands for the strain
and I follow order in which the enzyme was isolated.
Q. 6.* How does a restriction nuclease function? Explain.
Ans: Restriction nuclease cuts DNA at the specific locations, exonuclease cuts DNA at the ends
whereas; endonuclease cuts at specific position within the DNA.
Q. 7.* State the role of DNA ligase in biotechnology.
Ans: DNA ligase is a vital enzyme required for important cellular process such as DNA
replication, repair of damaged DNA and recombination. The enzyme mediates the formation of
phosphodiester bonds between adjacent 3’ hydroxyl and 5’ phosphate terminals.
Q. 8.* How can a bacterial DNA be released from the bacterial cell for biotechnology
experiments?
Ans: So as to extract or release the bacterial DNA from the cell, the bacterial cell should be treated
with the enzymes like lysozyme. The DNA is enclosed in the membrane. The membrane should
be broken so as to get DNA, and other macromolecules such as proteins, polysaccharides and
lipids.
Q. 9. What are the basic steps through which a genetically modified organism can be created?
Ans: There are three basic steps through which the genetically modified organism can be created,
they are: (i) Identification of a DNA that consists desirable genes. (ii) Introduction of the
identified DNA into host cells. (iii) Maintenance of introduced DNA in the host and transfer of
it in the next generation.
Q. 10.* 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.
Ans: The restriction enzyme cuts the DNA sequence away from the centre of the palindrome site,
in between the same two bases on the opposite strands, leaving a single stranded portion at the
end of the hanging stretches which are known as ‘Sticky ends’ on each of the strand. Thus, they
form hydrogen bonding between the cut counterparts. It also facilitates the action of ligase, an
enzyme so as to connect the foreign DNA with the vector.
Q. 11.* Why is ‘Plasmid’ an important tool in biotechnology experiments?
Ans: Plasmids play a role in expressing particular genes and they act as vectors for transferring
piece of foreign DNA attached to them.
Q. 12.* Why do fragments move towards the anode during gel electrophoresis?
Ans: In this procedure, the fragments of DNA being negatively charged, molecules get separated
by forcing them to move towards the anode under the electric field through the matrix.
Q. 13. * Why is it not possible for an alien DNA to become part of a chromosome anywhere
along its length and replicate normally?
Ans: This is because; along the DNA there is specific site from where the replication process starts.
This site is known as ‘ori’ or origin of replication. Hence, the alien DNA has to bind and start the
process of replication only from ori.
Q. 14.* Write the role of ‘Ori’ and ‘restriction’ site in a cloning vector pBR322.
Ans: It is the genetic sequence that acts as the initiation site for replication of DNA. Restriction
site is the recognition site for restriction enzyme such as EcoRI, Hind III etc. The recognition sites
are the genetic sequence from where the restriction enzymes cut the DNA molecule.
Q. 15.* Explain the work carried out by Cohen and Boyer that contributed immensely in
biotechnology.
Ans: These two scientists were successful in isolating the antibiotic resistant gene, from the
plasmid of a bacterium that was resistant to the antibiotic drug, and then linked this gene with
the plasmid of Salmonella typhimurium.
Q. 16.* List the key tools and steps used in recombinant DNA technology.
Ans: The key tools that are utilised in recombinant DNA technology are restriction enzymes,
polymerase and ligase, competent host organisms and the cloning vector.
The steps in recombinant DNA technology are:
(i) Isolation of DNA.
(ii) Fragmentation of DNA using restriction enzyme.
(iii) Isolation of desired DNA fragment.
(iv) Amplification of gene of interest.
(v) Ligation of the DNA fragments into a vector using DNA ligase.
(vi) Transfer of Recombinant DNA into the host cell.
(vii) Culturing the host cell on suitable medium on commercial scale.
(viii) Desired product is extracted.
(ix) Downstream processing of the product.
(x) Ready for marketing.
Q. 17* How is the action of normal endonuclease enzymes different from that of restriction
endonuclease?
Ans: Restriction endonuclease always recognizes specific nucleotides sequence within the DNA
whereas; normal endonuclease cut at random position within the DNA sequence.
Q. 18. What is gene gun?
Ans: It is the method of introducing an alien DNA into the host cells, in which the cells are
bombarded with the micro particles of gold or tungsten coated with DNA with a high velocity.
This method of transforming the recombinant DNA into host cell is known as biolistics or gene
gun.
Q. 19. Describe briefly the Bioreactor.
Ans: They are the large vessels used for the large-scale production of biotechnological products
extracted from raw materials. Such vessels provide suitable conditions to obtain the desired
product by providing the optimum temperature, pH, vitamin, oxygen, etc. Bioreactors ha ve an
oxygen delivery system, a foam control system, a pH, a temperature control system, and a
sampling port to obtain a small volume of culture for sampling.
Q. 20.* What is a primer? What is its role in PCR?
Ans: A primer is a small segment of DNA that binds to a complementary strand of DNA. Primers
are required for starting the functioning of DNA polymerase enzyme in the process of PCR.
Q. 21.* Mention the number of primers required in each cycle of polymerase chain reaction
(PCR). Write the role of primers and DNA polymerase in PCR.
Ans: Two sets of primers are required in the PCR. Primers are required for starting the
functioning of DNA polymerase enzyme in the process of PCR.
Q. 22.* How are DNA fragments visualised during gel-electrophoresis? What is elution?
Ans: To visualise the DNA, the gel is stained with a fluorescent dye that binds to the DNA, and
is placed on an ultraviolet transilluminator that shows up the stained DNA as bright bands. The
dye can also be mixed with the gel before it is poured. The DNA fragments glow, allowing to see
the DNA present at different locations along the length of the gel. In gel-electrophoresis, the
separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This
step is called elution.
Q. 23.* Explain the process of gel-electrophoresis technique.
Ans: Gel-Electrophoresis in an analytical separation proceduce used in molecular biology to
separate and identify molecules such as DNA, RNA, protein, complexes on the basis of size. The
separation is achieved by loading the sample in a gel made up of small pores and setting an
electric field across the gel. The molecules migrate across the gel based on the electric charge (i.e.,
negatively charged molecules move away from the negative pole) and smaller molecules move
faster than large molecules; thus, a size separation is achieved. The gel offers molecular sieving
effect and thus helps in separation of charged sample based on size.
Q. 24. What do you mean by Palindrome? Give some examples of Palindrome DNA sequences.
OR
*Explain palindromic nucleotide sequence with the help of suitable example.
Ans: A palindrome is a sequence of letters or words that reads forwards and backwards in the
similar fashion. Genetically, a palindrome is a sequence of nucleotides along the DNA or RNA
containing the same series of nitrogenous bases similar from both the directions. Examples of
such palindromes are given below:

Q. 25. What are the important steps of recombinant DNA technology?

Ans: The important steps of recombinant DNA technology are:
(i) Isolation of genetic material.
(ii) Cutting of DNA at specific locations.
(iii) Amplification of Gene of Interest using amplifying methods like PCR technique.
(iv) Insertion of recombinant DNA in the host cell.
(v) Gaining the foreign gene product.
Q. 26.* Illustrate the recognition sequence of EcoRI and mention what such sequences are
called? How does restriction endonuclease act on the DNA molecule?
Ans: EcoRI slices the DNA between the bases G and A only when the sequence GAATTC is
present in the DNA. Such sequences are known as palindromic sequences. It is the sequence of
base pairs that reads the same on the two strands on the condition that the orientation of reading
must be the same. The restriction endonuclease cuts the DNA at specific positions. It inspects
the length of a DNA. Once the enzyme finds the specific location, it gets bind and cuts each of
the two strands of double helix at the specific points in their sugar phosphate backbones.
Q. 27.* Name and describe the technique that helps in separating the DNA fragments formed
by the use of restriction endonuclease.
Ans: The method is gel electrophoresis. In this method the fragments of the DNA are allowed to
separate under the electric field which is passed through the matrix. The commonly used matrix
is agarose gel. As the DNA fragments have negative charge on them, they get separated by
forcing them to move towards the anode or the positive charge. These DNA fragments separate
on the basis of the sizes through the sieving effect that is provided by the agarose gel.
Q. 28. From what have you learnt, can you tell whether enzymes are bigger or DNA is bigger
in molecular size? How did you know?
Ans: Enzymes are smaller in size as compared to DNA molecules. It is because DNA contains
genetic information for the development and functioning of all living organisms. It contains
instructions for the synthesis of proteins and DNA molecules. Also, enzymes are proteins which
are synthesised from a small stretch of DNA known as ‘genes’, which are involved in the
generation of the polypeptide chain.
Q. 29.* EcoRI is a restriction endonuclease. How is it named so? Explain. Write the sequence
of DNA bases that the enzyme recognizes. Mention the point at which the enzyme makes the
cut in the DNA segment.
Ans: The nomenclature as EcoRI comes from the Escherichia coli RY 13. In Eco, the first letter
stands for the genus whereas the second two letters stands for the species of the prokaryotic cell.
Steward Linn and Werner Arber isolated for the first time the enzymes from E.coli which are
responsible for the restriction of the growth of the Bacteriophage. The recognition sequence of
this enzyme is a palindrome. A palindrome is a sequence of base pairs that can be read in a
similar way from both the ends of the DNA strand. EcoRI cuts the DNA between bases G and A
only when the sequence GAATTC is available in the DNA molecule.
Q. 30.* (i) Expand VNTR and describe its role in DNA fingerprinting. (ii) List any two
applications of DNA fingerprinting technique.
Ans: (i) VNTR—Variable Number of Tandem Repeats. It is used as a probe because of its high
degree of polymorphism.
(ii) (a) DNA fingerprinting is used for paternity testing through the use of PCR technique which
produces the genetic fingerprint and is highly specific for each individual.
(b) It is used in the Data security where DNA regions used for individual identification are
specific isolated genetic loci in the non-coding regions of the genomic DNA.
Q. 31.* Why do Iepidoplerans die when they feed on Bt cotton plant? Explain how does it
happen.
Ans: Bt cotton plant contains inactive toxin protein. Once the insect ingests it, the inactive
protoxins are converted into active form due to alkaline pH in the gut which solubilises the
crystals. The activated toxins bind to the surface of the midgut, creates prose, causes swelling,
lysis eventually leading to the death of the insect pest.
Q. 32.* During a fire in an auditorium a large number of assembled guests got burnt beyond
recognition. Suggest and describe a modern technique that can help hand over the dead to
their relatives.
Ans: DNA fingerprinting is the modern technique that can help the authorities to hand over the
dead to their relatives.
It includes series of steps which are as follows:
(i) Isolation and digestion of DNA by restriction endonuclease.
(ii) Separation of DNA fragments by electrophoresis and transferring them to synthetic
membranes such as the nitro-cellulose or nylon membrane.
(iii)Hybridisation using labelled probe.
(iv)Detection of hybridised DNA fragments by autoradiography.
(v) Matching banding pattern of DNA, DNA fingerprints or autoradiograms of the guests who
lost their lifes during the accident and that of their relatives.
Q. 33.* Explain enzyme-replacement therapy to treat adenosine deaminase deficiency.
Mention two disadvantages of this procedure.
Ans: Adenosine deaminase (ADA) deficiency is a genetic disorder. In this disease, the gene
coding for the enzyme ADA gets deleted leading to deficiency of ADA and problems in immune
system. Adenosine deaminase (ADA) deficiency in patients can be treated by gene replacement
therapy or enzyme replacement therapy. In this treatment, patients are regularly injected with
the functional ADA enzyme. Disadvantages of this procedure: (i) It does not completely
eradicate the disease. (ii) Requirement of repeated doses of the enzyme makes it expensive.
Q. 34.* What is a GMO? List any five possible advantages of a GMO to a farmer.
Ans: Those plants, bacteria, fungi or animals whose genes have been altered by manipulation are
called Genetically Modified Organisms (GMOs).
Advantages: (i) Tolerance to abiotic stresses like cold, drought, salt, heat etc.
(ii) Reduce reliance on chemical pesticides.
(iii)Reduced post harvest losses.
(iv) Increased efficiency of mineral usage by plants.
(v) Enhanced nutritional value.
(vi) To create tailor made plants.
Q. 35.* Suggest and describe a technique to obtain multiple copies of a gene of interest in vitro.
Ans: Polymerase Chain Reaction. (PCR) technique is used to obtain multiple copies of a gene of
interest. Multiple copies of the gene of interest is synthesised in vitro using two sets of primers
and enzyme DNA polymerase. The enzyme extends the primers using nucleotides provided and
genomic DNA as template. The process of DNA replication is repeated several times for
amplification of DNA with the help of thermostable DNA polymerase which remains active
during high temperature induced denaturation of double stranded DNA.
Q. 36.* Describe the formation of recombinant DNA by the action of EcoRI.
Ans: EcoRI identifics its palindromic sequence on both vector DNA and foreign DNA (GAATTC).
it cuts starands of DNA a little away from the centre of palindromic sites but between the same
two bases (G and A). This leave single stranded portion at the end (sticky ends) on each strand.
For recombination both vector DNA and foreign DNA with similar sticky ends are joined by the
enzyme DNA ligase.

Q. 37.* Describe the process of amplification of “gene of interest” using PCR technique.
Ans: The PCR technique begins with denaturation of desired DNA into two strands, each acting
as a template. For each strand, separate set of primers are used. DNA polymerase extends the
primers using nucleotides provided in the reaction. If the process of replication of DNA is
repeated many times, the segment of DNA can be amplified to approximately billion times. Such
repeated amplification is achieved by the use of a thermostable DNA polymerase which is
isolated from a bacterium Thermus aquaticus.
Denaturation—Double helical DNA is denatured by providing high temperature (95-degree
Celsius). DNA polymerase does not get degraded in such high temperature. The DNA
polymerase used in this reaction is thermostable and is isolated form the thermophilic bacteria.
Thermus aquaticus (Taq).
Annealing: It is the step in which primers are annealed to single stranded DNA templates. Two
sets of primers are used. The temperatures of the reaction mixture is lowered to 50-60ºC for some
seconds to allow annealing of primers. DNA polymerase extends the primer in 5’ to 3’ direction.
Extension—Replication of DNA occurs in vitro.
This cycle is repeated several times to generate up to 1 billion identical copies of the DNA.
Q. 38. Write a brief note on restriction enzyme. Explain the mechanism of restriction enzyme.
OR *Why are molecular scissors so called? Write their use in biotechnology.
Ans: The restriction enzymes act as molecular scissors that cut the DNA segment at the specific
site. The discovery of two such enzymes was made in 1963, one of which was identified later as
restriction endonuclease. The first restriction endonuclease was HIND II, the function of which
is to recognize the specific sequence of six base pairs and cut the segment at that location.
The restriction enzyme belongs to a larger class named nucleases. The two types of such enzymes
are endonuclease and exonucleases. The exonucleases play a role in removing the nucleotides
from the ends of DNA. It also inspects the length of DNA sequence. The endonuclease makes a
cut at specific positions of DNA fragment. Once the restriction endonuclease finds a specific
location, that has a capacity to recognize the palindromic nucleotide sequence; the enzyme gets
bind to DNA and makes a cut at specific points in sugar phosphate backbone of each of two
strands of the double helix. After the cutting, the overhanging sticky portion plays its role. The
sticky end allows joining of two counterparts of DNA strand with the help of another enzyme
called DNA ligase. The cuts made by restriction endonuclease give similar sticky ends at both
the strands. After cutting, separation and getting attached to the Host DNA, the recombinant
DNA is formed.
Q. 39.* Describe the characteristics a cloning vector must possess. Why the DNA cannot pass
through the cell membrane? Explain. How a bacterial cell is made ‘competent’ to take up
recombinant DNA from the medium?
Ans: The cloning vector must possess following characteristic features: (i) Origin of replication
(ORI): It is the initiation point from where the replication process starts and any piece of DNA
when linked to this sequence can be made to replicate the DNA within the host cell. The ORI is
also responsible for controlling the number of copies of the linked DNA. (ii) A selectable marker:
The vector must have a selectable marker which will help it in identifying and eliminating the
non-transformants and selectively permitting the growth of transformants only. (iii) It must
possess the cloning site or recognition site for a restriction enzyme to recognize. As DNA is a
hydrophilic molecule, it cannot pass through the cell membrane. The bacterial cell is made
competent through treating it with the specific concentration of Ca2+ ions. Further incubation of
the cells on the ice and providing short heat shocks can make the bacterial cell competent to take
up the recombinant DNA available in the medium.
Q. 40. Write a note on separation and isolation of DNA fragments through Gel Electrophoresis.
OR
*Explain the basis on which the gel electrophoresis technique works. Write any two ways the
products obtained through this technique can be utilised.
OR
*Name the technique used for separation of DNA fragments. Write the type of Matrix used in
the technique. How is the separated DNA visualised and extracted for use in recombinant
technology?
Ans: The process of cutting of DNA fragments by restriction endonuclease gives rise to the
formation of fragments of DNA. Such fragments of DNA can be separated by the laboratory
procedure called Gel Electrophoresis. The fragments of DNA to be separated are pumped into
the matrix of agarose gel. The entire matrix is exposed to an electric field. As the DNA fragments
are negatively charged, they tend to move towards the anode in the electric field. Thus the
fragments get separated on the basis of the sizes through the sieving effect that is provided by
the agarose gel. Smaller the size of the DNA fragment, farther it moves. After staining the DNA
fragments, the separation can be visualised. The staining of fragments is done by using a
compound named Ethidium bromide and then exposed to the ultra violet radiation. The
separated DNA fragments aren’t visible in the regular white light. In presence of UV light the
separated DNA fragments are orange in colour due to which Ethidium bromide can be seen.
Lastly, the process of elution is carried out. It is the process in which the separated bands of DNA
are cut out from the agarose gel and extracted from the gel piece. Now such purified DNA
fragments are ready for use to construct the recombinant DNA by using a cloning vector.
Q. 41. How are the following used in biotechnology:
(i) Plasmid DNA (ii) Recognition sequence *(iii) Gel electrophoresis
Ans: (i) Plasmid DNA: A plasmid is an extra-chromosomal, autonomously replicating circular
DNA. The plasmid DNA acts as the vectors to transfer the piece of DNA that is attached to it.
The construction of the first recombinant DNA molecule emerged from the possibility of linking
a gene encoding the antibiotic resistance with a native plasmid of Salmonella.
(ii) Recognition sequence: Hind II was the first restriction endonuclease. During its discovery it
was found that the Hind II used to cut the DNA molecules at a particular point by recognising a
specific sequence of six base pairs. This specific base sequence is known as the recognition
sequence for Hind II.
(iii)Gel Electrophoresis: It is the technique used to separate the DNA fragments after cutting the
DNA with the help of restriction endonuclease. The fragmentation is so complex that the
separation requires the process which is known as Gel Electrophoresis. In this technique the
natural tendency of the DNA fragments is utilised. As the fragments carry negative charge, they
tend to run towards the positive charge under the electric field.