PRINCIPLE AND PROCESSES

BIOTECHNOLOGY:

CHAPTER – 11

BIOTECHNOLOGY: PRINCIPLE AND PROCESSES

The technique of using life organisms or enzymes from This is because the alien piece of DNA has now become a part
organisms so as to produce products and processes that are of a chromosome which has the ability to replicate in a
useful to humans is called biotechnology. chromosome.
According to European Federation of biotechnology (EFB), There is a specific DNA sequence called the ‘origin of
Biotechnology is the integration of natural science and replication’ that is responsible for initiating the replication,
organisms, cells, parts thereof, and molecular analogs for so for the multiplication of any alien piece of DNA in an
products and services. organism, it needs to be a part of a chromosome that has a
specific sequence known as ‘origin of replication’.
Principles of Biotechnology
So alien DNA is linked with the ‘origin of replication’ so that
Biotechnology has two main techniques:
this alien piece of DNA can replicate and multiply itself in the
Genetic Engineering: Genetic engineering is described as host organism, it is also called as cloning.
the alteration of an organism's genome (DNA and RNA). It
Construction of an artificial rDNA molecule:
entails the introduction of additional genes into host species
• The potential of connecting an antibiotic resistance gene
in order to improve their function or characteristic, hence
with a native Plasmid of Salmonella typhimurium led to
altering the host organism's phenotype.
the creation of the first recombinant DNA.
Bioprocess engineering: Sterility is maintained in chemical • Stanley Cohen and Herbert Boyer extracted the
engineering processes to allow only desirable bacteria to antibiotic resistance gene by removing a fragment of DNA
thrive for the production of biotechnological goods such as from a Salmonella typhimurium plasmid (autonomously
antibiotics, vaccines, enzymes, and so on. reproducing circular extra-chromosomal DNA). The
The technique of genetic engineering includes: discovery of molecular scissors'–restriction enzymes–
o Creation of recombinant DNA made it feasible to cut DNA at specified spots.
o Gene cloning • The cut piece of DNA was linked with the plasmid DNA
o Gene transfer with the help of DNA ligase that acts on the cut ends of the
The fate of the piece of DNA transferred to alien DNA molecules and then joins their ends, this gives rise to
organism: the DNA transferred into the alien organism may rDNA.
not be able to multiply itself in the progeny cells of the • When this DNA is transferred into E.coli, it has the
organism but when it gets integrated into the genome of the potential to replicate numerous times utilising the new
recipient it may multiply and be inherited along with the host host DNA polymerase enzyme. Cloning of antibiotic
DNA. resistance gene in E.coli refers to the capacity to multiply
copies of an antibiotic resistance gene in E.coli.
• These “recombinant DNA” (rDNA) molecules are then Each restriction endonuclease functions by ‘inspecting’ the
introduced into host cells, where they can be propagated length of a DNA sequence. Once it finds its specific
and multiplied. recognition sequence, it will bind to the DNA and cut each of
the two strands of the double helix at specific points in their
ASSERTION AND REASON sugar -phosphate backbones. Restriction enzymes cut the
Q1. EFB stands for. strand of DNA a bit away from the palindrome site's centre
(a) Ethiopian Federation for Biotechnology between the identical two bases on opposing strands with
(b) European Federation for Biotechnology sticky strand. The strands' stickiness facilitates the operation
(c) Ecuador Federation for Biotechnology of the enzyme DNA ligase.
(d) None of the above
Restriction endonucleases are utilised in genetic engineering
S1. (b) to create recombinant DNA molecules made up of DNA from
Q2. Which of the following are also called as ‘molecular diverse sources or genomes.
scissors’? When the same restriction enzyme is used to cut the DNA,
(a) DNA polymerases the resulting fragments contain the same type of Sticky-
(b) Restriction endonucleases
ends, the stickiness of the ends facilitates the action of the
(c) RNA polymerases
(d) All of the above enzyme DNA ligase.

S2. (b)
Tools of Recombinant DNA technology
The major tools that are used in rDNA technology are:
• Enzymes: the major enzymes used in rDNA technology
are:
• Molecular scissors: these are the restriction enzymes,
that belong to the class Nucleases. They are of two
types:
(i) Endonucleases: they remove nucleotides from
somewhere within the DNA, it is very helpful in
producing specific cuts in the DNA. The specific base
sequence is of six base pairs.
Palindromes: Palindromes are group of letters that
form the same words when read both forward and
backward. E.g. “MALYALAM”.
(ii) Exonucleases: they remove nucleotides from the ends
of the DNA. E.g. Hind II. Each restriction endonuclease
is unique to a palindromic nucleotide sequence in the
DNA.
Today, we know more than 900 restriction enzymes Representation of recombinant DNA technology
that have been isolated from over 230 strains of Agarose gel Electrophoresis:
bacteria each of which recognise different recognition ✓ the cutting of DNA by restriction endonucleases results
sites. in the fragments of DNA fragments can be separated by a
technique known as gel electrophoresis.
✓ DNA is negatively charged and hence it can be separated
by making them move towards the anode under an
electric field through a matrix.
✓ the commonly used Matrix in DNA gel electrophoresis is
agarose that is a natural polymer extracted from
seaweeds.
✓ DNA fragments resolve according to the size through the
sieving effect provided by the agarose gel.
✓ the DNA fragments that are separated can be seen
after staining the DNA with ethidium Bromide and
later by exposing it to ultraviolet radiation.
✓ the separated DNA fragments can be seen as orange
colour bands that can be cut out from the gel and purified
Steps in the formation of DNA by the action of
from the gel, this process is called DNA elution.
restriction endonuclease enzyme EcoRI.
✓ the DNA fragments are used in constructing the also been disarmed and are now used to deliver
Recombinant DNA by attaching them with cloning desirable genes into animal cells.
vectors.
The main features a vector should have for gene
cloning are:
(i) Origin of replication (ori): When any fragment of DNA
is joined to this sequence, it may be made to reproduce
within the host cells. This segment is in charge of
regulating the copy number of the connected DNA.
(ii) Selectable marker: this assists in recognising and
removing non-transformants while selectively
A typical agarose gel electrophoresis allowing the development of transformants.
Transformation is the process of inserting a
o Polymerases: These enzymes catalyse the synthesis of fragment of DNA into a host bacteria. In general,
DNA molecules from nucleoside diphosphate and are genes encoding antibiotic resistance, such as
essential for DNA replication, they usually work in ampicillin, chloramphenicol, tetracycline, or
groups to create two identical DNA duplexes from a kanamycin, are thought to be helpful selection
single original DNA duplex, during this process DNA markers for E. coli.
polymerase reads the existing DNA strands to create two
news strands that match the existing one. (iii) Cloning sites: the vector must have a single recognition
o Ligases: these enzymes catalyse the joining of two large site for the generally used restriction enzymes for
molecules of DNA by forming a chemical bond. joining the foreign DNA, as multiple recognition sites
inside the vector may yield several fragments, that may
make the gene cloning process tricky. The foreign DNA
is ligated at a restriction point found in one of the two
antibiotic resistance genes.
Q1. Molecular scissors belong to which of the following
class of enzymes? e.g. E.coli cloning vector pBR322 shown in the diagram.
(a) Nucleases (b) Hydrolases
(c) Lyases (d) Ligases
S1. (a)
Q2. Which of the following is used to dye the DNA
fragments so as to view it under UV radiation?
(a) Methyl orange (b) Ethidium bromide
(c) Indigo (d) Malachite green
S2. (b)
• Vectors: Plasmids and Bacteriophages are two
typical vectors for cloning. They have the ability to
multiply within bacterial cells independently of
chromosomal DNA regulation. Bacteriophages have
very high copy numbers of their genome within
bacterial cells due to their large frequency per cell.
For example, Agrobacterium tumifaciens, a pathogen E.coli cloning vector pBR322
of several dicot plants is able to deliver a piece of DNA Insertional Inactivation: the cloned DNA fragment
known as ‘T-DNA’ to transform normal plant cells into disrupts the coding sequence of a gene, this is called
a tumor and direct these tumor cells to produce the insertional inactivation, the method is very important
chemicals required by the pathogen. The tumor in screening recombinants.
inducing (Ti) plasmid of Agrobacterium tumifaciens
has now been modified into a cloning vector which is e.g. Blue white selection:
no more pathogenic to the plants but is still able to use (i) The insertional inactivation of the lac Z gene contained
the mechanisms to deliver genes of our interest into a on the vector is the basis for this approach.
variety of plants (ii) The lac Z gene encodes the beta-galactosidase enzyme,
which may convert a chromogenic substrate into a blue
Similarly, retroviruses in animals have the ability to product.
transform normal cells into cancerous cells. They have
(iii) If this lac Z gene is inactivated by inserting a target ✓ Transforming the recombinant DNA into the host
DNA fragment into it, the formation of blue ✓ Culturing the host cells in a medium at large scale
colonies is stopped, and white colonies result. ✓ Extraction of the desired product:
Isolation of Genetic material: Enzymes such as
(iv) This allows us to distinguish between recombinant
lysozyme, cellulase, and chitinase are used to separate
(white) and non-recombinant (blue) colonies.
genetic material from other macromolecules (fungus).
• Host organisms: these are the bacterial cells that take
Spooling can be used to remove DNA that has
up the recombinant DNA, as DNA is hydrophilic, it
separated. RNA can be removed by using ribonuclease,
cannot pass through the cell membrane of bacteria,
whereas proteins may be removed by using protease.
thus the bacterial cells have to be made competent to
take up the DNA. This is done as follows: Cutting of DNA at specific location: To access the
course of a restriction enzyme digestion, restriction
(a) A simple chemical treatment with divalent calcium
enzyme and Agarose gel electrophoresis are used.
ions boosts the efficacy of host cells to take up the
After cutting the source and vector DNA with a
rDNA plasmids (through cell wall pores).
particular restriction enzyme to remove the 'gene of
(b) rDNA may also be turned into host cells by incubating interest' from the source DNA.
both on ice, then temporarily placing them at 42 degree
C (Heat Shock), and then returning to ice. This allows Amplification of gene of interest with PCR: here the
the bacteria to consume the recombinant DNA. gene of interest is amplified and multiple copies are
made with help of primers, polymerases and a set of
(c) Using a glass micropipette, rDNA is directly injected
primers.
into the nucleus of cells in the Microinjection
technique. Insertion of Foreign DNA into the host cell: there are
several methods of introducing the recombinant DNA
(d) Biolistics / Gene gun approach, which has been
into recipient cells, the recipient cells after making
created to transfer rDNA into plant cells primarily by
them competent to receive, take up DNA present in a
the use of a Gene / Particle gun. In this procedure,
surrounding, thus if a recombinant DNA bearing piece
minute gold/tungsten particles are coated with the
for resistance to an antibiotic (ampicillin) is
desired DNA and battered onto cells.
transferred into E.coli cells, the host cells become
(e) The last technique employs "Disarmed Pathogen" transformed into ampicillin-resistant cells, if we
Vectors (Agrobacterium tumefaciens), which, once spread the transformed cells on Agar plates containing
infected, transport the recombinant DNA into the host. ampicillin only transformants will grow and the
untransformants will die.

Q1. The features a vector should have for it to be perfect

for gene cloning is.
(a) Cloning sites (b) Selectable marker
(c) Ori (d) All of the above
S1. (d)
Q2. To make the bacterial cells competent, they are given
heat shock at a temperature.
(a) 41 degree (b) 42 degree
(c) 43 degree (d) 44 degree
S2. (b)
How does the Recombinant DNA technology works?
Various steps in recombinant DNA technology are as follows:
✓ Isolation of DNA
✓ Fragmentation of DNA by restriction endonuclease
✓ Isolation of desired DNA fragment
✓ Ligation of DNA fragment into the vector
Polymerase chain Reaction
Note: a thermostable DNA polymerase (isolated from a
bacterium, Thermus aquaticus), which remains active during
the high temperature induced denaturation of double
stranded DNA. Q1. In DNA purification protocol, the DNA ultimately
precipitates out during the procedure by the addition
After having cloned the gene of interest and having optimised
of pure.
the conditions to induce the expression of the target protein,
(a) Iso propyl alcohol (b) Ethanol
one has to consider producing it on a large scale.
(c) Methyl alcohol (d) None of the above
Small volume cultures cannot yield appreciable quantities of
S1. (b)
products. To produce in large quantities, the development of
bioreactors, where large volumes (100-1000 litres) of culture Q2. In PCR technique, annealing is the.
can be processed, was required. Thus, bioreactors can be (a) 1st step of the protocol
thought of as vessels in which raw materials are biologically (b) 2nd step of the protocol
converted into specific products, individual enzymes, etc., (c) 3rd step of the protocol
using microbial plant, animal or human cells. A bioreactor (d) 4th step of the protocol
provides the optimal conditions for achieving the desired
S2. (b)
product by providing optimum growth conditions
(temperature, pH, substrate, salts, vitamins, oxygen). Down Stream Processing
After completion of the biosynthetic stage, the product has to
The most common types of bioreactors used are of
be subjected through a series of processes before it is ready
stirring type.
for marketing as a finished product. The processes include
A stirred-tank reactor is usually cylindrical or with a curved separation and purification, which are collectively referred to
base to facilitate the mixing of the reactor contents. The as downstream processing. The product has to be formulated
stirrer facilitates even mixing and oxygen availability with suitable preservatives.
throughout the bioreactor.
 The use of biology to develop technologies and products for the welfare of human beings is known as biotechnology. The two
core techniques that give rise to modern biotechnology are genetic engineering and bioprocess engineering. Genetic
engineering allows the isolation and introduction of only the desired genes into the organism without introducing the
undesirable genes, main tools of genetic engineering are restriction enzymes, vectors, host.

The basic steps in genetic modification of an organism are the identification of desired DNA fragment, introduction of desired
DNA fragment into suitable host, maintaining foreign DNA in the host and its transfer to the progeny.

Agarose gel electrophoresis is the technique in which the DNA fragments obtained through restriction are separated.

To produce large quantities of recombinant protein, large vessels known as bioreactors are used. A bioreactor provides the
optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate,
salts, vitamins, oxygen).

The processes and methods involved in the separation and purification of the desired product are called as downstream
processing. In case of drugs, the product needs to be suitably formulated and drug tested before being made available
commercially.

The techniques of genetic engineering which include creation

The separated DNA fragments on agarose gel electrophoresis
of recombinant DNA, use of gene cloning and gene transfer.
can be viewed under UV light after staining it with an
In the year 1963, the two enzymes responsible for restricting intercalating agent called ethidium bromide that absorbs
the growth of bacteriophage in Escherichia coli were isolated light in the range of 260-330 nm.
called as restriction endonucleases.
Microinjection, biolistics and ‘disarmed pathogen’ vectors are
Today we know more than 900 restriction enzymes that have used to introduce alien DNA in the host cell.
been isolated from over 230 strains of bacteria. The most commonly used bioreactors in recombinant DNA
Restriction enzymes belong to a larger class of enzymes technology are are of stirring type.
called nucleases.
 MULTIPLE CHOICE QUESTIONS
Q1. Which of the following are part of biotechnology? Q8. What is gene transfer?
(a) In-vitro fertilisation leading to test tube baby (a) the introduction of new protein in an existing
(b) Synthesising a gene organism cell
(c) Developing a DNA vaccine (b) the introduction of new DNA in an existing
(d) All of the above organism cell
(c) the introduction of new RNA in an existing
Q2. Which of the following is the definition given by the organism cell
European Federation of biotechnology that (d) the introduction of chromosome in an existing
encompasses both traditional views and modern organism cell
molecular biotechnology?
(a) the innovation of natural science and organism’s Q9. What is a plasmid?
cells, parts thereof, and molecular analogues for (a) it is an autonomously replicating circular
products and services extrachromosomal DNA
(b) the integration of natural science and organism’s (b) it is an autonomously replicating linear
cells, parts thereof, and molecular analogue for extrachromosomal DNA
products and services (c) it is an autonomously replicating circular
(c) the inactivation of natural science and organisms chromosomal DNA
cells, parts thereof, and molecular analogue for (d) it is an autonomously replicating circular
products and services extrachromosomal RNA
(d) the integration of organisms cells, parts thereof, Q10. Who among the following accomplished the
and molecular analogue for products and services construction of the first component DNA in 1972?
Q3. Which of the following techniques enables the birth of (a) Von Bear
(b) Alexander Fleming
modern biotechnology?
(c) Stanley Cohen and Herbert Boyer
(a) Plant breeding
(d) Ernst Heckel
(b) Genetic engineering
(c) Bioprocess engineering Q11. Which of the following is also referred to as molecular
(d) Both (b) and (c) scissors?
(a) Polymerases (b) Restriction enzymes
Q4. What is genetic engineering? (c) Transcriptase (d) None of the above
(a) it is a technique to alter the chemistry of genetic
material DNA Q12. What is the use of vector in biotechnology?
(b) it is a technique to alter the chemistry of genetic (a) They help in the transfer of piece of RNA attached
material RNA to it
(c) it is a technique to alter the chemistry of genetic (b) They help in the transfer of piece of protein
material DNA and RNA attached to it
(d) it is a technique to alter the chemistry of genetic (c) They help in the transfer of piece of DNA attached
material protein to it
(d) They help in the transfer of piece of chromosome
Q5. Genetic engineering includes. attached to it
(a) Creation of recombinant DNA
(b) Gene cloning Q13. Which of the following enzyme at once cut DNA
(c) Gene transfer molecules and join their ends?
(d) All of the above (a) DNA polymerase
Q6. Which of the following is responsible for initiating (b) Restriction enzymes
replication in a chromosome? (c) DNA ligase
(a) Promoter (d) Reverse transcriptase
(b) Origin of replication Q14. Which of the following enzyme always cut DNA
(c) Plasmid molecules at a particular point by recognising a specific
(d) None of the above sequence of 6 base pairs?
(a) Bam H 1 (b) Eco R 1
Q7. What is cloning?
(c) Hind II (d) Hind III
(a) making single identical copy of any template DNA
(b) making multiple identical copies of any template Q15. How many restriction enzymes have been discovered
DNA till now?
(c) making multiple identical copies of any protein (a) More than 1000 (b) More than 900
(d) making multiple identical copies of chromosome (c) More than 800 (d) More 700
Q16. Which of the following is a source of Eco R 1? Q25. Which of the following metal is used for DNA in the host
(a) Escherichia coli RY11 (b) Escherichia coli RY12 cell through gene gun technology?
(c) Escherichia coli RY13 (d) Escherichia coli RY14 (i) Silver (ii) Gold
(iii) Tungsten (iv) Both (b) and (c)
Q17. Which among the following is the function of
exonucleases? Q26. Which of the following enzyme is used to treat the
(a) the remove nucleotides from the middle of the DNA bacterial cells for the isolation of DNA?
(b) the remove nucleotides from the ends of the DNA (a) Lysozyme (b) Cellulase
(c) the remove nucleotides from the template DNA (c) Chitinase (d) All of the above
(d) the remove nucleotides from newly processed DNA Q27. Which of the following can be used to remove RNA
Q18. Which of the following is correct about restriction during DNA isolation procedure?
endonucleases? (a) Proteases
(a) they recognise a specific nucleotide sequence in the (b) Ribonucleases
DNA (c) DNAses
(b) they recognise a specific palindromic sequence in (d) Restriction endonucleases
the DNA Q28. Which of the following is used to precipitate purified
(c) they recognise a specific palindromic nucleoside DNA during the procedure of DNA isolation?
sequence in the DNA (a) Chilled methanol (b) Chilled water
(d) they recognise a specific palindromic nucleotide (c) Chilled ethanol (d) Chilled buffer
sequences in the DNA
Q29. Which of the following is the correct form of PCR?
Q19. What is the charge on the DNA molecule? (a) Polymer Chain Reaction
(a) Positive charge (b) Pole Chain Reaction
(b) Neutral (c) Prime Chain Reaction
(c) Negative charge (d) Polymerase Chain Reaction
(d) None of the above Q30. What is a recombinant protein?
Q20. Name the matrix that is used to separate DNA (a) it is a protein encoding DNA that is expressed in a
fragments using gel electrophoresis. heterologous host
(a) Cellulose (b) Agar (b) it is a protein encoding gene that is expressed in a
(c) Agarose (d) Starch heterologous host
(c) it is a protein encoding RNA that is expressed in a
Q21. Which of the following compound is used to stain the heterologous host
separated DNA fragments to view them under the UV (d) it is a protein encoding ribosome that is expressed
radiation? in a heterologous host
(a) Methyl orange (b) Ethidium bromide
Q31. In gel electrophoresis, DNA fragments are separated on
(c) Methyl red (d) Naphthol green B
the basis of.
Q22. Which of the following is responsible for controlling (a) Charge (b) Size
the copy number of the linked DNA during replication? (c) Weight (d) All of the above
(a) Selectable marker Q32. Which of the following helps in the selection of
(b) Origin of replication transformed cells on a Petri dish?
(c) Cloning sites (a) Antibiotic resistance gene
(d) Promoter (b) Recognition sites
Q23. What is transformation? (c) Cloning sites
(a) it is a procedure through which a piece of RNA is (d) Ori C
introduced into a host bacterium Q33. Eco R1 is a restriction enzyme. In the name, what does
(b) it is a procedure through which a piece of protein is ‘co’ stands for?
introduced into a host bacterium (a) Colon (b) Coli
(c) it is a procedure through which a piece of (c) Coffee (d) Coenzyme
chromosome is introduced into a host bacterium Q34. Which of the following is the source of restriction
(d) it is a procedure through which a piece of DNA is endonucleases?
(a) Fungal cells (b) Bacterial cells
introduced into a host bacterium
(c) Plant cells (d) Virus
Q24. Which of the following diavalent cation is used to
Q35. After running gel electrophoresis, the extraction of
increase the efficiency of DNA so that it easily enters
DNA fragments from agarose gel is called as.
the bacterium through the pores present in its walls? (a) Cutting (b) Elution
(a) Magnesium (b) Barium (c) Separation (d) Ligation
(c) Calcium (d) All of the above
Q36. Which of the following is a protein digesting enzyme?
Q46. Look at the diagram given below and name ‘A’.
(a) Chitinase (b) Cellulase
(c) Protease (d) Lipase
Q37. Which of the following is correct about plasmid?
(a) It is bacteria
(b) It is extra chromosomal DNA
(c) It is a plant
(d) It is virus
Q38. What are bacteriophages?
(a) They are fungus that infect bacteria
(b) They are virus that infect bacteria
(c) They are protozoans that infect bacteria
(d) They are helminths that bacteria
Q39. What are basic steps of genetically modifying an
organism?
(a) identification of DNA with desirable genes
(b) introduction of the identified DNA into the host
(c) maintenance of introduce DNA in the host and
transfer the DNA to its progeny
(d) All of the above
(a) DNA ligae
Q40. Which of the following cannot be included under
(b) Reverse transcriptase
biotechnology? (c) Proteases
(a) Development of COVID vaccine (d) Taq polymerase
(b) Test tube baby technique
(c) Setting of curd Q47. What are the optimum growth conditions for large
(d) Treating defective gene scale production of cultures in a bioreactor?
(a) Temprature, oxygen (b) pH, vitamins
Q41. Which of the following is the correct full form of EFB? (c) Substrate and salts (d) All of the above
(a) European Federation of Biotechnology
Q48. What are palindromic sequences?
(b) Eastern Federation of Biotechnology (a) A sequence on ds DNA or RNA which if read in one
(c) Ethopian Federation of Biotechnology direction is identical to sequence in the same
(d) European Federation of Biology direction on the complementary strand also.
Q42. What is the meaning of annealing in PCR technique? (b) A sequence on only ds DNA which if read in one
(a) It is the joining of RNA primers to the template direction is identical to sequence in the same
DNA direction on the complementary strand also.
(b) It is the joining of DNA primers to the template (c) A sequence on ds RNA which if read in one direction
DNA is identical to sequence in the same direction on
(c) It is the joining of protein to the template DNA the complementary strand also.
(d) It is the joining of DNA primers RNA (d) A sequence on protein if read in one direction is
identical to sequence in the same direction on the
Q43. Taq polymerase is isolated from. complementary strand also.
(a) Thermus aquaticaus
Q49. Which of the following is not a restriction enzyme?
(b) Aspergillus niger
(a) Bam H1 (b) Hind III
(c) Clostridium butylicum (c) Eco RI (d) Lipase
(d) None of the above Q50. Sticky ends of DNA fragments during recombinant DNA
Q44. What is the number of steps in each cycle of PCR? technology can be joined by.
(a) 1 (b) 2 (a) DNA polymerase (b) RNA polymerase
(c) 3 (d) 4 (c) DNA ligase (d) Taq polymerase

Q45. What is denaturation of DNA? ASSERTION AND REASON

(a) It is the separation of ss DNA into strands
(b) It is the separation of ds RNA into single strands Direction: in the following questions, a statement of
(c) It is the separation of ss RNA into single strands assertion (A) is followed by a statement of reason (R). Choose
(d) It is the separation of ds DNA into single strands the correct option among a, b, c and d.
 (b) Both assertion (A) and reason (R) are true but
Q1. Assertion (A): DNA ligase plays an important role in
reason (R) is not the correct explanation of
recombinant DNA technology.
assertion (A)
Reason (R): The linking of antibiotic resistance gene
(c) Assertion (A) is true but reason(R) is false
with plasmid vector became possible by enzyme DNA
(d) Assertion (A) is false but reason(R) is true
ligase.
(a) Both assertion (A) and reason (R) are true and Q4. Assertion(A): The selection of recombinants due to
reason (R) is the correct explanation of assertion the inactivation of antibiotics is a cumbersome
(A) procedure.
(b) Both assertion (A) and reason (R) are true but Reason (R): It requires simultaneous plating on two
reason (R) is not the correct explanation of plates having different antibiotic’s.
assertion (A) (a) Both assertion (A) and reason (R) are true and
(c) Assertion (A) is true but reason(R) is false reason (R) is the correct explanation of assertion
(d) Assertion (A) is false but reason(R) is true (A)
(b) Both assertion (A) and reason (R) are true but
Q2. Assertion (A): Restriction enzymes belong to a larger
reason (R) is not the correct explanation of
classes of enzymes called nucleases.
assertion (A)
Reason (R): Each restriction enzyme recognises a
(c) Assertion (A) is true but reason(R) is false
specific palindromic sequence in the DNA.
(d) Assertion (A) is false but reason(R) is true
(a) Both assertion (A) and reason (R) are true and
reason (R) is the correct explanation of assertion
(A)
TRUE AND FALSE
(b) Both assertion (A) and reason (R) are true but
reason (R) is not the correct explanation of Q1. DNA is a hydrphobic molecule.
assertion (A) Q2. In order to make the bacterial cells competent, they
(c) Assertion (A) is true but reason(R) is false are treated with a specific concentration of a divalent
(d) Assertion (A) is false but reason(R) is true cation, such as calcium, which increases the efficiency
Q3. Assertion (A): During the gel electrophoresis, the with which DNA enters the bacterium through pores in
fragments of DNA move towards the anode. its cell wall.
Reason (R): DNA fragments are negatively charged Q3. In micro-injection, recombinant DNA is directly
molecules. injected into the cytoplasm of an animal cell.
(a) Both assertion (A) and reason (R) are true and
reason (R) is the correct explanation of assertion Q4. Biotechnology deals with large scale production and
(A) marketing of products and processes using live
organisms, cells or enzymes.

PRACTICE QUESTIONS
Q1. Which of the following tools of recombinant DNA cells are spread on agar plates containing ampicillin,
technology is incorrectly paired with its use? then.
(a) restriction enzyme - Production of RFLPs (a) both transformed and untransformed recipient
(b) DNA ligase-that cuts DNA, creating the sticky ends cells will die
(c) DNA polymerase - used in a polymerase chain (b) both transformed and untrasformed recipient cell
reaction to amplify section of DNA will be grow
(d) reverse transcriptase - production of cDNA from (c) tranformed recipient cells will grow and
mRNA untransformed recipient cells will die
(d) transformed recipient cells will die and
Q2. In r-DNA technology or genetic engineering elution
untransformed recipient cells will grow
means.
Q4. The technique that serves the purpose of early
(a) Remove the DNA from centrifuge tube after
diagnosis of disease or pathogen.
centrifugation
(a) Recombinant DNA technology
(b) The separated band of DNA are cut out from the gel
(b) Polymerase chain reaction
and extracted from the gel piece
(c) Enzyme linked immuno sorbent assay
(c) Separation of the recombinant protein from
(d) All the above
recombinant cell
(d) Insertion of recombinant DNA into the host cell Q5. To denature the DNA template in PCR it is heated to.
(a) 70°C (b) 54°C
Q3. If a recombinant DNA bearing gene for ampicillin
(c) 80°C (d) 94°C
resistance if transferred into E.Coli cells and the host
Q6. Most common matrix is agarose a natural polymer (b) Restriction exonuclease + DNA polymerase
used in gel electrophoresis is extracted from. (c) Alkaline phosphate + DNA Ligase
(a) an animal (b) a fungus (d) RNA polymerase + DNA polymerase
(c) Sea weeds (d) None of these
Q16. In the vector pBR322 there is.
Q7. If the bacterium does not have any insert, then in the (a) One selectable marker
presence of chromogenic substrate, it gives. (b) Two selectable markers
(a) Red coloured colonies (c) Three selectable markers
(b) Colourless colonies (d) None of the above
(c) Blue colonies
Q17. The enzymes responsible for restricting the growth of
(d) Green colonies
bacteriophage in E-coli were isolated in 1963, these
Q8. Insertional inactivation results into inactivation of enzyme are.
which enzyme? (a) DNA ligases
(a) Transacetylase (b) Permease (b) Alkaline phosphatases
(c) Taq polymerase (d) -galactosidase (c) DNA polymerases
(d) Restriction endonuclease
Q9. The sequence which is responsible for controlling the
copy number of the linked DNA is. Q18. During the process of gene amplification using PCR, if
(a) Coding sequence very high temperature is not maintained in the
(b) Promoter sequence beginning, then which of the following steps of PCR will
(c) Terminator sequence be affected first?
(d) Ori (a) Annealing (b) Extension
(c) Denaturation (d) Ligation
Q10. If any protein encoding gene is expressed in a hetero
Q19. DNA strands on a gel stained with ethidium bromide
logous host then protein is known as.
when viewed under UV radiation, appear as.
(a) Recombinant gene
(a) Yellow bands (b) Bright orange bands
(b) Recombinant protein
(c) Dark red bands (d) Bright blue bands
(c) Selectable marker
(d) Homogenous protein Q20. Which of the following is a correct sequence of steps in
a PCR (Polymerase Chain Reaction)?
Q11. Group of letters that form the same words when read
(a) Denaturation, Annealing, Extension
both forward and backward is called.
(b) Denaturation, Extension, Annealing
(a) Palindrome (b) Same words
(c) Extension, Denaturation, Annealing
(c) Opposite words (d) None of the above
(d) Annealing, Denaturation, Extension
Q12. The enzymes, which remove nucleotides from the ends
Q21. The laws and rules to prevent unauthorised
of the DNA are.
exploitation of bio-resources are termed as.
(a) Exonuclease (b) Endonuclease
(a) Biopatenting (b) Bioethics
(c) Cellulase (d) Hydrolase
(c) Bioengineering (d) Biopiracy
Q13. When the isolation of genetic material is done the RNA
Q22. In Recombinant DNA technology antibiotics are used.
can be removed by treatment with.
(a) to keep medium bacteria-free
(a) Protease
(b) to detect alien DNA
(b) Chitinase
(c) to impart disease-resistance to the host plant
(c) Ribonuclease
(d) as selectable markers
(d) Deoxyribonuclease
Q23. In a mixture, DNA fragments are separated by.
Q14. Roman numbers following the names of restriction
(a) Bioprocess engineering
endonuclease indicate.
(b) Restriction digestion
(a) The order in which the enzymes were isolated
(c) Electrophoresis
from that strain of bacteria
(d) Polymerase chain reaction
(b) strain of bacteria
(c) the order in which genus is taken to isolate the Q24. Identify the wrong statement with regard to
enzyme Restriction Enzymes.
(d) none of the above (a) Sticky ends can be joined by using DNA ligases.
(b) Each restriction enzyme functions by inspecting the
Q15. Which one of the following is must in Genetic
length of a DNA sequence.
engineering?
(c) They cut the strand of DNA at palindromic sites.
(a) Restriction endonuclease + DNA ligase +
(d) They are useful in genetic engineering.
polymerases
Q25. A selectable marker is used to. (b) Both assertion (A) and reason (R) are true but
(a) help in eliminating the non-transformants, so that reason (R) is not the correct explanation of
the transformants can be regenerated assertion (A)
(b) identify the gene for a desired trait in an alien (c) Assertion (A) is true but reason(R) is false
organism (d) Assertion (A) is false but reason(R) is true
(c) select a suitable vector for transformation in a
Q2. Assertion (A): In a chromosome there is a specific
specific crop
DNA sequence called the origin of replication, which is
(d) mark a gene on a chromosome for isolation using
responsible for initiating replication.
restriction enzyme
Reason (R): The construction of the first recombinant
Q26. A gene whose expression helps to identify transformed
DNA emerged from the possibility of linking a gene
cell is known as.
encoding antibiotic resistance with a native plasmid of
(a) Vector (b) Plasmid
Salmonella typhimurium.
(c) Structural gene (d) Selectable marker
(a) Both assertion (A) and reason (R) are true and
Q27. Which of the following is a restriction endonuclease? reason (R) is the correct explanation of assertion
(a) Hind II (b) Protease (A)
(c) DNase I (d) RNase (b) Both assertion (A) and reason (R) are true but
reason (R) is not the correct explanation of
Q28. Which vector can clone only a small fragment of DNA?
assertion (A)
(a) Bacterial artificial chromosome
(c) Assertion (A) is true but reason(R) is false
(b) Yeast artificial chromosome
(d) Assertion (A) is false but reason(R) is true
(c) Plasmid
(d) Cosmid Q3. Assertion (A): Endonucleases remove nucleotides
from the ends of the DNA.
Q29. Biolistics (gene-gun) is suitable for.
Reason (R): Each restriction endonuclease functions
(a) Constructing recombinant DNA by joining with
by ‘inspecting’ the length of a DNA sequence.
vectors
(a) Both assertion (A) and reason (R) are true and
(b) DNA finger printing
reason (R) is the correct explanation of assertion
(c) Disarming pathogen vectors
(A)
(d) Transformation of plants cells
(b) Both assertion (A) and reason (R) are true but
Q30. For transformation, micro-particles coated with DNA reason (R) is not the correct explanation of
to be bombarded with gene gun are made up of. assertion (A)
(a) Silicon or Platinum (b) Gold or Tungsten (c) Assertion (A) is true but reason(R) is false
(c) Silver or platinum (d) Platinum or zinc (d) Assertion (A) is false but reason(R) is true
Q4. Assertion (A): Transformation is a procedure through
ASSERTION AND REASON which a piece of DNA is introduced in a host bacterium.
Reason (R): Normally, the genes encoding resistance
Direction: in the following questions, a statement of to antibiotics such as ampicillin, chloramphenicol,
assertion (A) is followed by a statement of reason (R). Choose tetracycline or kanamycin, etc., are considered useful
the correct option among a, b, c and d. selectable markers for E. coli.
Q1. Assertion (A): Ethidium bromide helps in visualising (a) Both assertion (A) and reason (R) are true and
DNA in UV light only. reason (R) is the correct explanation of assertion
Reason (R): The dye absorbs light in the range of 260- (A)
330nm (b) Both assertion (A) and reason (R) are true but
(a) Both assertion (A) and reason (R) are true and reason (R) is not the correct explanation of
reason (R) is the correct explanation of assertion assertion (A)
(A) (c) Assertion (A) is true but reason(R) is false
(d) Assertion (A) is false but reason(R) is true

SOLUTIONS MULTIPLE CHOICE

S1. (d) Biotechnology deals with techniques of using live S3. (d) Genetic engineering and Biotechnology
organisms and enzymes from organisms to engineering are the basis of the origin of modern
produce products and processes are useful to biotechnology
humans S4. (c) Genetic engineering alters the genetic material
S2. (b) many definitions of biotechnology have been DNA or RNA and then it is introduced into the
proposed but this definition encompasses both host organism so as to change the phenotype of
traditional view and the modern molecular the host organism
biotechnology view also
S5. (d) Genetic engineering allows us to isolate and S19. (c) the charge on DNA is negative, this property is
introduce only a set of desirable genes without widely used to separate DNA fragments using gel
introducing the undesirable genes into the target electrophoresis technique
organism S20. (c) agarose is a natural polymer extracted from
S6. (b) Origin of replication is a specific DNA sequence on seaweeds
the chromosome that is responsible for initiating S21. (b) after staining the separated DNA fragments with
replication ethidium bromide, one can see orange colour
bands of DNA under UV light
S7. (b) Cloning is done when an alien DNA is linked with
the origin of replication so that this is alien piece S22. (b) origin of replication is a sequence from where
of DNA can replicate and multiply itself in the host replication starts, any piece of DNA when linked
organism to the sequence can be made to replicate within
the host cell, it also controls the copy number of
S8. (b) Gene transfer is an important aspect of genetic the linked-to DNA
engineering
S23. (d) transformation is a procedure of introducing a
S9. (a) A plasmid was used for the construction of the novel piece of DNA inside the host cell
first recombinant DNA by combining it with a
S24. (c) Diavalent cation like calcium increases the
gene encoding antibiotic resistance
porosity of the bacterial wall and introduces the
S10. (c) Stanley Cohen and Herbert Boyer accomplished r-DNA into the bacterial cell
the construction of first recombinant DNA by S25. (d) In gene gun, DNA particles are coated in the gold
isolating the antibiotic resistance gene by cutting or tungsten and then injected directly in the host
out a piece of DNA from a plasmid which was body.
responsible for conferring antibiotic resistance
S26. (d) lysozyme is obtained from bacterial cells
S11. (c) The discovery of restriction enzyme made the chitinase from fungal cells and cellulase from
cutting of DNA at specific location very easy plants
S12. (c) Just as a mosquito acts as an insect vector to S27. (b) DNA is interwined with protein, histones. RNA is
transfer the malarial parasite into the human also present that can be removed with the help of
body in the same way vectors like plasmid in enzyme ribonuclease
biotechnology help in the transfer of a piece of S28. (c) chilled ethanol easily precipitates the purified
DNA attached to it DNA out of the test tube
S29. (d) polymerase chain reaction is a reaction in which
S13. (c) Linking of any antibiotic resistance gene with the multiple copies of a gene of interest is synthesized
plasmid vector becomes easy with the help of in vitro using two sets of primers and the enzyme
enzyme DNA ligase that has a property to join cut DNA polymerase
ends of DNA S30. (b) the desired protein can be extracted from the
cloned genes and can be used for any purpose
S14. (c) Hind II was the first restriction endonuclease to
be discovered and it always cuts DNA molecules a S31. (b) in gel electrophoresis the DNA fragments resolve
particular point by recognising 6 place face according to the size due to the sieving effect
sequence provided by the agrose gel

S15. (b) till now more than 900 restriction enzymes have S32. (a) antibiotic resistance genes act as a marker on the
vector that helps in selection of the transformed
been isolated from over 230 strains of bacteria
cells
each of which recognise different recognition
S33. (b) Eco R 1 the letter R is derived from the name of
sequences the strain and ‘co’ stands for coli
S16. (c) Eco R 1 come Sfrom Escherichia coli RY11 S34. (b) in the 1963, two enzymes responsible for
S17. (b) exonucleases belong to the class of enzyme restricting the growth of bacteriophages in E.coli
nucleases and remove nucleotides from the ends were isolated. Later more research was made and
of the DNA were found useful, so they were commercially
isolated from bacterial cells for research
S18. (d) restriction endonucleases are very specific in purposes.
their function, once it finds it specific points in the
S35. (b) the separated bands of DNA are cut out from the
sequence it binds to the DNA and cut each of the
agarose gel and extracted from the gel piece, this
two strands of the double helix in the sugar
step is called as elution
phosphate backbone
S36. (c) protein can be easily removed from DNA by S46. (d) Taq polymerase helps in the extension of the
treating it with an enzyme called proteases primers using deoxynucleotides.
S37. (b) Plasmid is autonomously replicating unit present
S47. (d) a bioreactor is used to produce large volumes of
in bacterial cells
cultures to obtain the desired protein for the
S38. (b) Bacterial cells can also be infected by pathogens, desired product
like virus as they infect humans and plants, such
S48. (a) palindromic sequences are very important in
virus is called bacteriophages
biotechnology, the restriction enzymes cut the
S39. (d) All the above 3 are the 3 basic steps of genetically
strand of DNA a little away from the centre of the
modifying an organism.
palindromic site but between the same two bases
S40. (c) biotechnology is a restricted sense today it is used on the opposite strands
to refer to such processes which use genetically
S49. (d)
modified organisms to achieve the same on a
larger scale S50. (c) DNA ligases effectively joins the sticky ends of
DNA during r-DNA technology.
S41. (a) the European Federation of biotechnology has
given a definition of biotechnology that
encompasses both traditional view and modern
ASSERTION AND REASON
molecular biotechnology
S1. (a)
S42. (b) Annealing during PCR is achieved when the
temperature is lowered in the machine as per the S2. (b)
set protocol. S3. (a)
S43. (a) taq polymerase is a thermostable DNA S4. (a)
polymerase that is isolated from the bacterium
Thermus aquaticus and remains active during the TRUE AND FALSE
high temperature induced denaturation of double
stranded DNA S1. (False) DNA is a hydrophilic molecule.
S44. (c) Each cycle of PCR has three steps denaturation, S2. (True)
primer annealing and extension of primers
S3. (False) In micro-injection, recombinant DNA is
S45. (d) denaturation is the first step of PCR, in this the directly injected into the nucleus of an animal
double stranded DNA opens and converts into cell.
single strand so that the primers can anneal
S4. (True)

PRACTICE SOLUTIONS
S1. (b) Restriction enzymes cut DNA creating sticky ends of S5. (d) During this stage the cocktail containing the
restriction fragments. While DNA ligase joins these template DNA and all the other core ingredients
sticky ends to form recombinant DNA. is heated to 94-95⁰C.
S2. (b) cutting and extraction of DNA is done to S6. (c) the most commonly used matrix is agarose
separate the DNA from the gel in which it is which is a natural polymer extracted from
collected. Water or a low salt buffer is added to seaweeds.
break the cation bridge and dislodge the DNA
S7. (c) the presence of a chromogenic substrate gives blue
from the gel and elute it.
coloured colonies if the plasmid in the bacteria does
S3. (c) The cloning vector requires the presence of a not have an insert. Presence of insert results into
selectable marker, which helps in identifying and insertional inactivation of the -galactosidase and the
eliminating non-transformants and selectively colonies do not produce any colour, these are
permitting the growth of the transformants. identified as recombinant colonies.
Normally, the genes encoding resistance to
S8. (d) he presence of a chromogenic substrate gives blue
antibiotics such as ampicillin, chloramphenicol,
coloured colonies if the plasmid in the bacteria does
tetracycline or kanamycin, etc. are considered useful
not have an insert. Presence of insert results into
selectable markers for E. coli. The normal E.coli cells
insertional inactivation of the -galactosidase and the
do not carry resistance against any of these
colonies do not produce any colour, these are
antibiotics.
identified as recombinant colonies.
S4. (d)
S9. (d) Ori site or the site of origin controls replication in survive on antibiotic rich medium. Such genes that
circular plasmid DNA and hence, the copy number of the host cell requires for growth under certain
linked DNA in the vector. conditions and differentiate the recombinant cells
from non-recombinant ones are called as selectable
S10. (b) the protein is called a recombinant protein.
markers.
S11. (a) such group of letters are called palindrome. S23. (c) in electrophoresis, the negatively charged DNA
moves from negative terminal to the positive
S12. (a) exonucleases are the enzymes that are
terminal.
necessary for proofreading and removing the
nucleotides from the DNA ends and the S24. (a) Sticky ends contain free or hanging or unpaired
endonucleases are the enzymes that cut DNA at nitrogen bases which can pair to complementary
specific positions. bases present on other DNA segment required to
create a recombinant DNA. A ligase is required in
S13. (c) removal of RNA can be done by ribonucleases
absence of sticky ends to join together two segments
and proteins can be done by protease.
of DNA.
S14. (a) The Roman numerals are used to identify specific
S25. (a) Only those cells which have been transformed
enzymes from bacteria that contain multiple
successfully can be traced by marker. So, this helps
restriction enzymes. Typically, the Roman numeral
in eliminating non-transformants and permitting
indicates the order in which restriction enzymes
transformants.
were discovered in a particular strain.
S15. (a) Key tools for recombinant DNA technology are S26. (d) In recombinant DNA technology, selectable
restriction endonuclease enzyme and DNA ligases markers are the specific genes that are used to
for recombinant DNA production, vectors that help identify the transformants from the non-
in carrying and integrating the desired gene, host transformants after the process of
organism is that one in which recombinant DNA is recombination. These genes are used to detect
introduced, polymerase enzymes whether the incorporation of a nucleic acid
sequence has been successful into an organism's
S16. (b) In the cloning vector pBR322, ampicillin and
DNA.
tetracycline resistance genes are the selectable
markers. The role they play is that they help in the S27. (a) A restriction enzyme or restriction endonuclease
selection of transformed cells from non transformed is an enzyme that cleaves DNA into fragments at or
cells. They also help distinguish recombinant cells near specific recognition sites within the molecule
from non-recombinant cells. known as restriction sites. Restrictions enzymes are
one class of the broader endonuclease group of
S17. (d) they were restriction endonucleases.
enzymes.
S18. (c) the denaturation step will be affected.
S28. (c) Plasmids are autonomously replicating circular
S19. (b) the DNA stained with ethidium bromide when extra-chromosomal DNA. They are the standard
viewed under UV light appear bright orange. cloning vectors and the ones most commonly used.
Most general plasmids may be used to clone DNA
S20. (a) Polymerase chain reaction or PCR consists of the
insert of up to 15 kb in size.
following three steps:
Denaturation- The two DNA strands of template S29. (d) The biolistic method is a method of transformation
DNA separate from each other when heated to 92℃, of plants. It is used to transfer foreign DNA into a
next is annealing- The primers anneal to the 3’ end plant cell. For the purpose, cells are bombarded with
of single strands of DNA and the last is extension- high-velocity micro-particles of gold or tungsten
The primers are extended by DNA polymerase by coated with DNA.
the addition of nucleotides to form complete strands S30. (b)
of DNA. Hence the sequence of steps is denaturation,
annealing, extension. ASSERTION AND REASON
S21. (a) a patent is a, intellectual property right that
provides the owner the legal right to ensure S1. (a)
that no one else benefits from making, using or S2. (b)
selling an invention.
S3. (d) Exonucleases remove nucleotides from the
S22. (d) Vectors carry an antibiotic-resistant gene which ends of the DNA.
helps to select the recombinant cells from non-
recombinant ones as only recombinant cells would S4. (b)
exhibit the antibiotic resistance and would be able to