Biotechnology NEW

BIOLOGY-XII BIOTECHNOLOGY 1
BIOTECHNOLOGY
PRINCIPLES AND PROCESSES
Biotechnology is use of microorganisms, plant, animal cells, their components or enzymes

from organisms to produce products and processes use for human welfare.
 Term biotechnology was given by Engineer - Karl Ereky (1917) for large scale production of pigs.
 European Federation of Biotechnology (EFB) - Biotechnology is integration of natural science
and organisms, cells, parts there of and molecular analogues for products and services.
 US National science Foundation - Controlled use of biological agents, such as
microorganism or cellular components, for beneficial use.
 British Biotechnologist – Application of biological organism, system or processes to
manufacturing and service industries.
DNA manipulation biotechnology or recombinant DNA technology or genetic engineering
 Gene manipulation is a science concern with development of recombinant DNA. It is named as
DNA manipulation biotechnology or recombinant DNA technology or genetic engineering.
 This technology involves cutting and pasting of desired DNA, based on two important
discoveries in bacteria -
(i) Plasmids in bacteria, which can undergo replication independent of chromosomal DNA.
(ii) Restriction endonucleases (molecular scissors), which break DNA at specific sites. (Arber,
Nathan and Smith-1970)
 Father of genetic engineering is Paul Berg. He introduce gene SV-40 into bacterium by
lambda phage virus.
Construction of first artificial recombinant DNA
 Cohen and Boyer (1973) first of all introducing a piece of antibiotic resistance gene into
plasmid of Salmonella typhimurium. This modified plasmid was inserted into E. coli to get
clones of recombinant DNA.
 As female Anopheles acts as vector to transfer malarial parasite into human. Similarly plasmid
functioning as vector to carry piece of DNA into host organism.
Old or Traditional Biotechnology – It includes processes that are based on natural capabilities
of microorganisms. It is also known as conventional technology.
 Curd, vinegar, ghee, wine, beer and other alcoholic beverages, idli, dosa, cheese, paneer and
some other foods have been produced using traditional biotechnology.
 In Indian, Ayurveda, production of ‘Asva’ ‘Arista’ is done through traditional biotechnology.
Traditional biotechnology is called art rather than science.
Modern Biotechnology - Human insulin is produced by a transgenic E. coli. Proteins produced
by transgenes are called recombinant proteins. Production technologies based on genetic
engineering is known as modern biotechnology. It developed during 1970.
ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

Recombinant DNA technology – It includes formation of recombinant DNA and introduction of
rDNA into appropriate host. rDNA is formed by combining DNA from different organisms.
Concept and Principles of Genetic Engineering
 Sexual reproduction is base of concept of genetic engineering. It is more useful than asexual
reproduction because it gives rise variations and new combinations.
 Variations are very important to organisms as well as population while asexual reproduction
preserves genetic characters.
 By traditional hybridization methods in plant and animal breeding number of undesirable genes
with desired genes introduce but genetic engineering provide only desire genes.
 A piece of DNA would not be able to multiply itself in organism while it gets incorporated into
genetic material of recipient. After incorporation DNA has become part of a chromosome and
now possesses ability to replicate.
 When alien DNA link with origin of replication site get ability for replication and multiply
itself in host organism. It is refer as cloning (form multiple identical copies of template DNA).
Thus genetic engineering is alternately called recombinant DNA technology or gene cloning.
Principles of Modern Biotechnology - Two main techniques that form modern biotechnology are –
(i) Genetic Engineering – Techniques change chemistry of genetic material and alter phenotype
of host organism.
(ii) Chemical Engineering - It maintain microbial contamination free for manufacturing of
biotechnological products like antibiotics, vaccines, enzymes, medicines, hormones, etc.
Tools of Recombinant DNA Technology
(A) Enzymes (B) Cloning vectors (Vehicle DNA) (C) Competent host
(A) Enzymes – In genetic engineering different kinds of specific enzymes are used –
(1) Lysing Enzymes - These enzymes are used to get DNA for genetic experiments. Lysozyme is
used to bacterial cell wall. In plant cell, cell wall is dissolve by cellulase, while in fungi; it is
dissolve by chitinase.
(2) Restriction Enzymes or molecular Scissors - These enzymes are used to break DNA
molecules also called nucleases. Restriction enzymes are of three types —
1. Exonucleases 2. Endonucleases 3. Restriction endonucleases
(a) Exonucleases - They remove nucleotides from terminal ends (either 5’ or 3’) of DNA in
one strand of duplex.
(b) Endonucleases - They cut at specific position within DNA. These enzymes do not cleave
ends and involve only one strand of DNA duplex at a time.
Exonucleases Endonucleases
1. These cleave base pairs of DNA at their terminal 1. They cleave DNA at any point except terminal ends.
ends.
2. They act on single strand of DNA or gaps in 2. They cleave one strand Fig. (a) or both stands Fig. (b)
double stranded DNA. of double stranded DNA.
3. They do not cut RNA. 3. They may cut RNA.

(c) Restriction endonucleases - Restriction
enclonuclease was discovered by Arber (1962) in
bacteria. They act as “molecular scissors” or ‘chemical
scalpels’.
 They recognize base sequence at palindrome sites and
cut them. Example - EcoRI of E. coli, recognizes base
sequence GAATTC in DNA duplex and cuts its strands
between G and A as shown below –
5’— G A AT T C —3’
3’ — CT TA A G —5’
Types of Restriction Endonucleases
Type-I
 These enzymes consist of 3 different subunits.
 They require ATP, Mg+2 and S-adenosyl methionine for restriction.
 Type-I recognize specific sites within DNA but do not cut these sites.
 They are not used in recombinant DNA technology.
Type-II
 These enzymes are simple and require Mg+2 ions for restriction.
 Only type II restriction enzymes are used in recombinant DNA technology.
 They can be used in vitro to recognize and cut within specific DNA sequence typically
consisting of 4 to 8 nucleotides.
Type-III
 These enzymes consist of two different subunits.
 They require ATP, Mg+2 and S-adenosyl methionine for restriction.
 They recognize specific sites within DNA but do not cut these sites.
 These restriction endonucleases are not used in recombinant DNA technology.
 They have intermediate properties between type I and type II.
Restriction-Modification System (Werner Arber)
 Restriction Modification System functions as defense system mechanism in many bacteria.
This system was explained by Arber in 1965. Restriction-Modification system consists of
two components –
(i) Primarily, Restriction enzyme identifies foreign DNA or viral DNA and cuts into pieces by
restriction endonucleases.
(ii) Secondly, modification enzyme adds CH3 group ‘within’ bacterial DNA and modify them due
to that it is not recognized by restriction enzyme. Now, modified DNA is fail to recognize by
restriction enzyme and not cut that DNA.
 First restriction endonuclease discovered was Hind II. It was isolated from Haemophilus
infiuenzae Rd. Hind II always cut DNA molecules at particular point at specific sequence of
six base pairs. It produces blunt ends.
 Today know more than 900 restriction enzymes that have been isolated from over 230 bacteria.
 Restriction endonuclease recognizes specific sequence; it binds to DNA and cuts each of two
strands of double helix in their sugar phosphate back bones.

 Palindromic sequences - Special sequence in DNA recognized and cut by restriction
endonuclease is called palindromic sequence. Palindromes are groups of letters that form same
words when read in both directions forward and backward. Examples – MADAM,
MADAMADAM and MALAYALAM. Following sequences read same on two strands in 5’—
> 3’ direction. This is also true when we read in 3’—> 5’ direction.
5’—GA AT TC—3’
3’—CT TA AG —5’
Sticky ends or cohesive ends
 Restriction enzymes cut DNA strand
little away from centre of palindrome
sites but between same two bases of
opposite strands.
 This leaves single stranded unpaired
bases at cut ends. These ends with
unpaired bases are called sticky ends
or cohesive ends.
 Sticky ends are named because they
form hydrogen bonds with their
complementary cut counter parts.
Sticky ends facilitate action of
enzyme DNA ligase.
Nomenclature of Restriction Enzymes
 Restriction enzymes (Type II) are named by bacterium from they have been isolated.
1. First letter used for enzyme is first letter of bacterium’s genus name (in italics),
2. Then two letters of its species (italics),
3. Strain of organism
4. Roman numeral signifying order of discovery.
 Enzyme Eco RI was isolated from bacterium Escherichia coli (Eco) strain RY 13 (R) and it
was first endonuclease (I) isolated. Discovery of these enzymes led to Nobel Prize for Arber,
Smith and Nathans in Medicine or Physiology in 1978.
Restriction enzymes type II, their source, recognition sequence and site of cleavage
Recognition Sequence and
Restriction Enzyme Source Product
Site of Cleavage
5’-A-G- C-T-3’
1. Alu I Arthrobacter luteus 3’-T-C- G-A-5’
5’-G-G-A-T-C-C-3’
Bacillus
2. Bam HI 3’-C-C-T-A-G-G-5’
amyloliquefaciens H
5’-G-A-A-T-T-C-3’
3. Eco RI (first isolated Escherichia coli RY13
Restriction endonuclease)
3’-C-T-T-A-A-G-5’

5 - C-C-T-G-G-3’
4. Eco RII Escherichia coli R245 3’-G-G-A-C-C -5’
Haomophilus 5’-G-G- C-C-3’

5. Hae III
aegyptius 3’-C-C- G-G-5’
5’-A-A-G-C-T-T-3’
Haemophiius
6. Hind III 3’-T-T-C-G-A-A-5’
intluenzae Rd
7. Hind II 5’-G-T-C’-G-A-C-3’
Haemophilus
(first discovered Restriction 3’-C-A-G -C-T-G-5’
endonuclease)
influenzae Rd
5’-G-T-C-G-A-C-3’
8. Sal I Streptomyces albus 3’-C-A-G-C-T-G-5’
Streptomyces 5’-A-G-T- A-C-T-3’

9. Sca I
caespitosus 3’-T-C-A- T-G-A-5’
5 -C-C-C- G-G-G-3’
10. Sma I Serratia marcescens
3’-G-G-G- C-C-C-5’
(3) DNA Ligases (Joining or Sealing Enzymes)
 These enzymes functioning as “genetic gum” and form phosphor-di-ester bonds between
adjacent nucleosides as well as covalent linkage between two stranded of DNA.
 Ligase enzyme requires phosphate group at 5’carbon and hydroxyl group at 3’carbon of
adjacent nucleoside to from phosphor-di-ester bond.
 These enzymes help in sealing gaps in DNA fragments. Hence, act as molecular glue.
 This enzyme used in rDNA technology in phase T4-DNA-ligase and discovered by Har
Gobind Khorana.
(4) Alkaline Phosphatase (AP)
 For ligation, phosphate group present at 5’ end. If phosphate group is removed, DNA cannot be
ligated. Enzyme alkaline phosphatase remove phosphate group from 5’ end and leaving a free
5-hydroxyl group. This enzyme isolated from bacteria (BAP) or calf intestine (CAP).
 It prevents unwanted self-ligation of vector DNA in procedures of rDNA technology.
(5) Synthesizing Enzymes
 These enzymes participate in synthesis of DNA strands. These are of two types –
1. Reverse transcriptase 2. DNA polymerase
(a) Reverse Transcriptase - This enzyme synthesize complementary DNA (cDNA) by using
mRNA as a template. It is useful in synthesis of cDNA and construction of cDNA clone bank.
(b) DNA Polymerase - This enzyme synthesis complementary DNA (cDNA). It works in 5’ 3’
and 3’ 5’ direction.
 DNA polymerase was discovered by Kornberg in E. coli (1956). Now it is known as DNA
polymerase I (DNA Pol.I). Other two enzymes are DNA polymerase II and DNA polymerase
III. These have almost similar catalytic activity.
 DNA Pol III is more active than other two in presence of ATP.

(B) Cloning Vectors (Vehicle DNA)
 Vectors are DNA molecules that carry a foreign DNA and replicate inside host cell.
 Vectors may be plasmids, bacteriophage, cosmids, phagemids, Yeast artificial chromosomes
(YACs), Bacterial artificial chromosomes (BACs), transposons or virus.
 Some shuttle vectors are also use; but plasmid and bacteriophage are commonly used vectors.
(1) Plasmid Vectors or natural vector
 Plasmids were discovered by William Hays and Joshua Lederberg (1952).
 These are extra-chromosomal, self—replicating, circular, double-stranded DNA, found
naturally in many bacteria and some yeast.
 Plasmids are not essential for normal cell growth and division, they concern with some special
traits. Eg. Resistance again certain antibiotics or toxins due to plasmid.
 Plasmid present as 1 or 2 copies or multiple copies (500—700) inside host organism.
ARTIFICIAL VECTOR OR pBR322 Vector - This
is first artificial cloning vector formed in
1977 by Boliver and Rodriguez.
Representation in pBR 322 plasmid is as
follows –
P - Plasmid
BR - Bolivar and Rodriguez
322- Number given to distinguish this
plasmid from others developed in same
laboratory. Example, there are plasmids
pBR324, pBR325, pBR328, pBR345 etc.
Structure of pBR 322 plasmid
 Following regions are present –
(i) Origin of replication (Ori) – Site of replication which allows production of multiple copies per cell.
r
(ii) Antibiotic resistance genes - there are two site as Ampicillin resistance (amp ) gene and
tetracycline resistance (tetr) gene in pBR322.
(iii) Recognition sites for restriction endonucleases - pBR 322 possess a variety of recognition sites for
restriction endonucleases.
r
 In amp gene Pst I and Pvu 1 are located.
r
 In tet gene BamHI, Sal I, are located.
 Some other restriction sites are EcoRI, Cla I, Hind III, Pvu II.
 rop codes for proteins involved in replication of plasmid.
Functioning - Presence of restriction sites within markers tetr and ampr permits an easy
selection for cells transformed with recombinant pBR322.
 Insertion of DNA fragment into plasmid using enzyme Pst I or Pvu I places DNA insert within
gene ampr; this makes ampr nonfunctional. Bacterial cells containing recombinant pBR322 will
be unable to grow in presence of ampicillin, but will grow on tetracycline.
 Similarly, when restriction enzyme Barn HI or Sal I is used, DNA insert is placed within gene
tet making it nonfunctional. Bacterial cells possessing recombinant pBR322 will grow on
ampicillin but not on tetracyclin.

(2) Bacteriophage Vectors
 Bacteriophages are viruses that infect bacterial cells. Injected DNA replicates with in bacteria
and form number of virus which burst cell (lytic cycle) and re-infect neighboring cells.
 This ability to transfer DNA from virus to bacteria during process of bacterial infection gave
scientists idea that specially designed virus vectors would be useful tools for gene cloning
experiments. For that lambda and M13 phase modified for development of cloning vectors.
(a) Lambda Phage Vector - It has a double-stranded, linear DNA genome of 5l4bp, in which 12
bases at each end are unpaired but complementary. These ends are sticky and are referred to as
cos-sites (cohesive end sites). These sites are important for packaging DNA into phage heads.
 An important feature of lambda genome is that a large fragment in central region of its genome
is not essential for lytic infection of E. coli cells. Therefore, vectors have been designed such
that this region can be replaced by a foreign DNA. These vectors allow cloning of DNA
fragments up to 23 kb in size.
(b) M13 Phage Vector – It is phage which infects E. coli having F-pilli. Its genome is a single
stranded, circular DNA of 6407 bp. Foreign DNA can be inserted into it without disrupting
any of essential genes.
 After entry in bacterial cell, it is converted into double-stranded as replicative form or RF,
which replicates until 100 copies are form. Now DNA replication becomes asymmetric and
single-stranded as M13 particles. RF is purified and manipulated like plasmid.
(3) Cosmid (Cos+plasmid) Vectors - Cosmids constructed by combining certain features of
plasmid and ‘cos’ sites of phage lambda. Simplest cosmid vector contains a plasmid origin of
replication, a selectable marker, suitable restriction enzyme sites and lambda ‘cos’ site.
Cosmids can be used to clone DNA fragments upto 45 kb in length.
(4) Phagemid Vectors - Phagemid is a composite structure made of bacteriophage and plasmid.
They are used for carrying larger DNA sequences.
(5) Bacterial Artificial Chromosome (BAC) Vectors - These are vectors based on natural, extra-
chromosomal plasmid of E. coli—Fertility or F-Plasmid or Fasmid.
 A BAC vector contains genes for replication and maintenance of F-factor, a selectable marker
and cloning site. These vectors can accommodate upto 300-350 kb of foreign DNA and are
also being used in genome sequencing projects.
(6) Yeast Artificial Chromosome (YAC) Vectors - These are used to clone DNA fragments of
more than 1 Mb in size, hence, they participate in mapping large genomes, e.g., Human
Genome Project.
 These vectors contain telomeric sequence, centromere and autonomously replicating sequence
from yeast chromosomes. They also contain restriction enzyme sites and genes which act as
selectable markers in yeast.
Differences Between BAC and YAC
BAC YAC
1. It is bacterial artificial chromosome. 1. It is yeast artificial chromosome.
2. Circular in shape. 2. Linear in shape.
3. It has genes for replication and maintenance of F- 3. It has telomeres, centromere and site for origin
factor, a selectable marker and cloning site. of replication,
4. Upper limit of foreign DNA to be inserted in BAC 4. Upper limit of foreign DNA to be inserted in
is about 300-3500 Kbp. YAC is about 2500-10000 Kbp* (kilobase pairs).

BIOLOGY – XII BIOTECHANOLOGY 8
(7) Animal and Plant Viral Vectors - Viruses that infect plant and animal cells have been manipulated
and used to introduce foreign DNA into plant and animal cells in culture.
 The natural ability of these viruses to adsorb to cells and infect them has been exploited to design
vectors to introduce foreign genes into eukaryotic cells in culture.
 Simian Virus 40 (SV-40) was used in first cloning experiment involving mammalian cells in
1979. Now, number of vectors based viruses like Adenovirus and Papilloma virus has been used
to clone genes in mammals.
 At present, retroviral vectors are most commonly used vectors in mammalian cells.
 In case of plants, plant viruses like Cauliflower Mosaic Viruses, Tobacco Mosaic Virus and
Gemini viruses were used but with limited success.
(8) Transposons as vectors - DNA sequences which change their location in genome are called
transposons. They are able to exercise from one locus and become inserted at separate locus.
They are used as vectors. Examples - Activator (Ac) and dissociation (Ds) are popular
transposons of Maize, are also called Ac—Ds Elements. Transposons of Drosophila are known
as P-Elements.
(9) Shuttle vectors - Plasmid vectors can replicate both eukaryotic cell and E. coli. Such vectors are
known as shuttle vectors. These vectors contain two types of origin of replication and selectable
marker genes, one type functions in eukaryotic cell and other functions in E. coli. Example -
Yeast episomal plasmid Yep.
 In plants, a naturally occurring plasmid of bacterium Agrobacterium tumefaciens called Ti-
plasmid has been suitably modified to function as a shuttle vector.
Characteristics of a Cloning Vector
(1) Origin of Replication (Ori)- This is sequence from where replication starts and any piece of
foreign DNA is linked to this sequence.
(2) Selectable Marker - Vector also requires a selectable marker (antibiotic resistance gene) to
identify and eliminate non-transformants and selectively permit growth of transformants.
 Transformation is a process through which a piece of DNA is introduced in a host bacterium.
As genes encoding resistance to antibodies like tetracycline, ampicillin etc. are useful in
selectable markers for E. coli. The common E. coli are not resistant against these antibiotics.
(3) Recognition Sites (Cloning sites) - Vector must have one unique restriction endonuclease
recognition site to insertion of foreign DNA. Unique restriction site allows particular enzyme to
cut vector only once.
 Most of used vectors contain unique recognition sites for several restriction enzymes in a small
region of DNA; it is refer as polylinker or multiple cloning site (MCS). Polylinker provides
flexibility in choice of restriction enzyme(s).
 The joining of foreign DNA is carried out at a restriction site located in one of two antibiotic
resistances.
 Example – when a foreign DNA linked at Bam HI site of tetracycline resistance gene in
pBR322; it lose their resistance ability for tetracycline but still grow in ampicillin medium.
 Non-recombinants grow on medium containing both antibiotics.
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CONTACT ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.
 In this example, one antibiotic resistance gene helps in selecting transformants but other
antibiotic resistance gene becomes inactivated due to insertion of foreign DNA and helps in
selection of recombinants.
Colour Reaction – To identify recombinant and non-recombinant colour reaction use in presence
of chromogenic substance. A recombinant DNA is inserted in coding sequence of an enzyme β-
galactosidase. This causes inactivation of enzyme, It is called insertional inactivation. If
plasmid in bacterium does not have an insert DNA, presence of a chromogenic substrate gives
blue coloured colonies. Presence of insert DNA results into insertional inactivation of β-
galactosidase enzyme and, therefore, bacterial colonies donot produce any colour. These
colonies are marked as recombinant colonies.
β-galactosidase, also called lactase, beta-gal or β-gal, is a glycoside-hydrolase-enzyme that catalyzes hydrolysis of β-
galactosides into monosaccharides through the breaking of a glycosidic bond. β-galactosides include carbohydrates containing
galactose where the glycosidic bond lies above the galactose molecule.
(4) Vectors for Cloning Genes in Plants and Animals – Today we know the procedure of
transferring genes into plants and animals from bacteria and viruses.
Example - Agrobacterium tumefaciens, (pathogen) of several dicot plants is able to transfer a
piece of DNA known as ‘T-DNA’ to convert normal plant cells into tumour and direct these
tumour cells to secrete chemicals required by pathogen.
 Similarly retroviruses (cause leukosis or sarcoma cancer) in animals and humans are able to
change normal cells into cancerous cells.
 Plasmid of Agrobacterium tumefaciens has been modified into cloning vector which is not
pathogenic to plants; it is still able to deliver genes of our interest into various plants.
 Similarly retroviruses are used to carry desirable genes into animal cells.
 Thus once a gene or DNA fragment is joined to a suitable vector it is transferred into a bacterial
plant or animal host where it undergoes multiplication.
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(C) Competent Host (For Transformation with Recombinant DNA)
 Transformation is a process by which a cell takes up naked DNA fragment from environment,
incorporates it into its own chromosomal DNA and finally expresses trait controlled by incoming
DNA. Competent host is essential for transformation with recombinant DNA.
 Many kinds of host cells - E.coli, yeast, animal and plant cells are available for genetic
engineering and kind of host cell used mainly depends on aim of cloning experiment.
 For expression of some eukaryotic proteins, eukaryotic cells may be preferred hosts. Yeasts have
been used extensively for functional expression of eukaryotic genes because they offer several
advantages. Yeasts are simplest eukaryotic organisms and like bacteria are single-celled,
genetically well-characterized, easy to grow and manipulate. They can be grown readily in both
small culture vessels and large scale bioreactors.
 Plant and animal cells may also be used as hosts in gene manipulation experiments and for
protein expression either in tissue culture or as cells in the whole organism to create genetically
modified plants and animals.
 DNA is a hydrophilic molecule; it cannot pass through membranes, so bacterial cells must be
made capable to take up DNA. This is done by treating them with a specific concentration of a
divalent cation – like Calcium which increases efficiency with which DNA enters bacterium
through pores in its cell wall.
 Recombinant DNA can then be forced into such cells by incubating cells with recombinant DNA
on ice, followed by placing them at 42°C (heat shock), and then putting them back on ice. This
enables bacteria to take up recombinant DNA.
 In animals term transformation is replaced by term transfaction.
Vector-less Gene Transfer - Different methods are use to introduce rDNA into recipient cells
without involving carrier molecules. Some of methods are described below –
1. Microinjection - In this method foreign DNA is directly
injected into nucleus of animal or plant cell by using micro
needles or micro pipettes. It is used in oocytes, eggs and
embryo.
Jeffey Chamberlain (U.S.A) has cured mice that inherited
a neuromuscular disease by this method which similar like
muscular dystrophy of humans.
2. Electro-poration - In this method electrical impulses induce
transient (temporary) pores in plant cell membrane through
which DNA molecules are incorporated.
3. Direct DNA Injection - Direct injection of DNA into skeletal muscle led to possibility of using
gene as vaccines. Due to low level of expression therapeutic benefits for treatment of genetic
disorder could not be derived. This method gave birth to concept of DNA vaccine or genetic
immunization.
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4. Gene Gun or Biolistic - Gene gun (Particle bombardment gun) is also available for vector-less
direct gene transfer. DNA coated on microscopic pellets of gold or tungsten of size 1-2 um is shot
with high velocity into target cells.
 Initially this technique develop for plants but now it is also
used for animal (tissue repair particularly cancer of mouth)
near wounds leading to reduction of healing time.
 This method is not useful in treatment of genetic disorder
but made great impact in field of vaccine development.
Gel Electrophoresis (A. Tiselius) (Separation and Isolation of
DNA Fragments) - After cutting of DNA by restriction
enzymes, fragments of DNA are formed. These fragments
separated by technique gel electrophoresis.
 Electrophoresis is a technique of separation of charged
molecules under influence of electrical field; +ve charged
molecules move towards cathode (-ve electrode) and -ve
charged molecules towards anode (+ve electrode) through
medium matrix. Matrix is agarose (polysaccharide extracted
from sea weeds).
 DNA fragments separate according to size of pores of
agarose gel. Hence smaller fragment move farther. Pore size
depends on agarose concentration.
 Agarose dissolves in hot water when this solution is cooled,
double helices filament form gel.
 DNA fragments seen after stain with ethidium bromide
(EtBr) followed by exposure to UV radiation as bright
orange coloured bands.
 Separated bands of DNA are cut out from agarose gel and
extracted from gel piece; it is known as elution. These
purified DNA fragments are used in formation of rDNA by
linking them with cloning vectors.
Processes of Recombinant DNA Technology
1. Isolation of DNA - DNA has to be isolated in pure form for
action of restriction enzymes.
 DNA can be released from cells by digesting cell envelope
by use of enzymes like lysozyme for bacterial cells.
Chitinase for fungal cells and cellulase for plant cells
 DNA is intertwined with histone proteins and RNAs. Proteins are removed by treatment with
proteases and RNAs by ribonucleases. Other impurities are removed by employing suitable
treatments. Purified DNA is precipitated by addition of chilled ethanol: it is seen as fine threads
in suspension.
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2. Cutting of DNA at Specific Locations - Fragmentation of DNA is carried out by incubating purified
DNA molecules with suitable restriction enzymes at optimal conditions of temperature and pH.
Agarose gel electrophoresis is used to check progress of restriction enzyme digestion.
 DNA is a negatively charged molecule, it moves towards +ve electrode (anode). After cutting
of DNA and vector DNA with a specific restriction enzyme, cut out ‘gene of interest’ from
source DNA and cut vector with space are mixed and ligase enzyme is added. This results in
preparation of rDNA.
3. Amplification of Gene of Interest by PCR - Amplification refers to process of making multiple
copies of DNA segment in vitro. It is done by polymerase chain reaction (PCR) process was
designed by K. Mullis. This technique involves three main steps -
1. Denaturation 2. Primer annealing 3. Extension of primers
a. Denaturation - Double-stranded DNA is denatured by using high temperature (94° to 96°C).
b. Primer annealing - Two sets of primers are used; primers are chemically synthesized short
segments of RNA (Oligo-nucleotides) that are complementary to segment of DNA (of interest).
This step is carried out at low temperature (40° to 60°C) depending on length and sequence of
primers.
c. Primer Extension (Polymerisation) - Taq
DNA polymerase (thermophilic bacterium
Thurmus acquaticus) synthesizes DNA
region between primers, using DNTPs (de-
oxy-nucleoside tri-phosphates) and Mg+2.
It means primers are extended towards
each other so that DNA segment lying
between two primers is copied. Optimum
temperature for this polymerization step is
72°C.
 For second cycle, DNA is again heated to
convert all newly synthesized DNA into
single strands, each of which can now
serve as a template for synthesis of more
new DNA. Thus extension product of one
cycle can serve as a template for
subsequent cycles and each cycle
essentially doubles amount of DNA from
previous cycle. As a result, from a single
template molecule, it is possible to
generate 2 molecules after n number of
cycles (2n).
 Basic requirements of a PCR reaction are following -
1. DNA Template - Any source that contains one or more target DNA to be amplified require.
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2. Primers – Primers (oligo-nucleotides, usually 10—18 nucleotides long), hybridize target DNA
in each strand of double helix. Two primers are required and these primers are oriented with
their ends facing each other allowing synthesis of DNA towards one another.
3. Enzyme - DNA polymerase (stable at high temperatures > 90°C) is required for synthesis of new
DNA. Polymerase which is generally used in PCR reactions is known as Taq polymerase
(Isolated from a bacterium Thermus aquaticus).
Application of PCR
a) Diagnosis of Pathogens - To detect pathogens, techniques based on detecting specific enzymes
or antibodies against disease-related proteins. But these techniques cannot be used for detecting
infectious agents that are difficult to culture or that persist at very low levels in infected cells.
 To overcome these problems, PCR-based assays have been developed that detect presence of
gene sequences of infectious agents. In recent times, PCR is being used in detection of HIV at an
early stage when infant is 18 months old.
b) Diagnosis of Specific Mutation - Mutations is genetic diseases. PCR is a valuable tool in
locating genetic basis of diseases. By using PCR phenylketonuria, muscular dystrophy, sickle
cell anaemia, AIDS, hepatitis, chlamydia and tuberculosis can be diagnosed.
c) DNA Fingerprinting - PCR is of immense value in generating abundant amount of DNA for
analysis in DNA fingerprinting technique used in forensic science to link a suspect’s DNA to
DNA recovered at a crime scene.
d) Detection of Specific Microorganisms - PCR is also useful in detection of specific
microorganisms from environment samples of soil, sediments and water.
e) In Prenatal Diagnosis - It is useful to detect genetic disease in foetus before birth. If disease is
not curable, abortion is recommended.
f) Diagnosis of Plant Pathogens - Many diseases of plants can be detected by using PCR.
Examples - viroids (associated with apple, grape, citrus, pear, etc.), viruses (like TMV, bean
yellow mosaic virus etc), bacteria, mycoplasma, etc.
g) In Paleontology - PCR is used to clone DNA fragments from mummified remains of humans
and extinct animals like wooly mammoth and dinosaurs.
h) Gene Therapy - PCR helps in monitoring a gene in gene therapy experiments.
Comparison between PCR and Gene Cloning
Parameter PCR Gene cloning

1. Efficient More Less
2. Requirement DNA Restriction enzyme, ligase, vector, bacterial cell.
3. Manipulation in vitro in vitro and in vivo
4. Cost Less More
5. Automation Yes No
6. Error probability Less More
7. Labour intensive No Yes
8. Time for a typical experiment 4 hours 2—4 days
9. User’s skill Less required More required
10. Application More Less
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It is expected that PCR will take over most of application of gene cloning
4. Insertion of rDNA into Host - Vector DNA (plasmid or foreign DNA) carrying gene of interest,
cut by restriction endonuclease. This process of cutting DNA by restriction enzymes is called
restriction digestion. With help of DNA ligase, complementary sticky ends of two DNAs are
joined (annealing) to produce a recombinant (chimera) DNA. Ligase forms new sugar-
phosphate bonds to join two DNAs.
 Eukaryotic genes do not function properly when cloned inside bacterial cell, because of their inability to
excise introns of eukaryotic genes and their destruction by bacterial restriction enzymes.
 In this condition, DNA is made from mRNA by reverse transcription or synthesized artificially. This
rDNA is inserted into host bacterium by transformation using cold CaCl2 solution.
 Bacterial cell containing rDNA of antibiotic is cultured in medium. It is then multiplied to get
clones of rDNA. These clones are isolated and desirable recombinant protein is obtained.
5. Obtaining desirable Gene Product - rDNA
is transferred into host DNA is multiplied
and start expression for desirable protein.
 After cloning of gene of interest maintain
optimum conditions to expression of target
protein and consider producing it on a
large scale.
 Any protein encoding gene is expressed in
heterologous host is known as
“recombinant protein”. Cells with genes
of interest grow on a small scale in
laboratory.
 To get large biomass of desire protein
bioreactors are use.
 In bioreactor medium is passed out from
one side and fresh medium is added from
other side to maintain cells in their
physiologically most active lag-
exponential phase (Lag phase—no
significant increase of cells; exponential
phase— rapid multiplication of cells).
Bioreactors (Fermenters)
 To produce large quantities of products,
bioreactor was required where large volume
(100-1000 litres) of culture can be
processed.
 Bioreactor provides optimal conditions for obtaining desired product like temperature, pH,
substrate, vitamins, oxygen and salts.
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 Bioreactors are vessels in which raw materials are biologically converted into specific products
by microbes, plant and animal cells and/or their enzymes.
 Commonly use bioreactors are of stirring type. Bioreactor has batch culture or continuous
culture. In continuous culture - culture medium is added and product is taken.
Stirred-tank bioreactor - Large stainless steel vessel with following parts –
(i) Cooling Jacket - Reduces temperature. Cooling jacket is provided with cooling water inlet and
cooling water outlet.
(ii) Air Inlet and Filter - Most fermentation are aerobic, requiring a large volume of sterile air.
(iii) Stirrer - It consist of a vertical rotating stirrer shaft and flat and vertical stirrer blades.
(iv) Sparger - It is a porous ring at bottom of tank for proper aeration.
(v) Lines (Ports) - Top of tank has a number of inlet tubes called lines through which materials can
be introduced or withdrawn.
(a) Inoculum Line (sample line) - Inoculum (modified microorganisms) added through this line.
(b) Antifoam Line - Introducing antifoaming agents.
(c) Nutrient Line - Introducing nutrients.
(d) pH Line - It is meant for introducing acid or alkali to maintain optimum pH.
(e) Dissolved Oxygen Line. Introducing dissolved oxygen.
(vi) Harvest Line (Product outlet). At the
base of tank, harvest line to extract
culture medium and products.
(viii) Temperature sensor and control unit -
To record temperature and control small
unit is present in bioreactor.
Fermentation Process
 Fermentation processes take place in
bioreactors. Term fermentations applied
only to anaerobic processes but now
include all processes whether aerobic or
anaerobic.
 All operations are carried out under sterile
conditions to avoid contamination of
culture.
 The product is either cells themselves (biomass) or some useful cell product. In batch
fermentations nutrients and microorganism are put in a closed reactor. Once inside fermenter,
organism grows and multiplies, using nutrient. When nutrients are utilized, product is separated
from microorganisms.
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Steps involved in Fermentation Process
(i) Isolation and Screening of Microorganisms - Industrially important microbes (bacteria, fungi,
algae) are isolated from soil, lakes and river mud. The desired microbes are screened and a pure
culture of selected microbes is developed.
(ii) Selection of nutrient Medium - Raw material like molasses, corn sugar, starch soyabean meal,
vegetable oils, etc. is selected for nutrient medium preparation.
(iii) Sterilization - Sterilized to remove or inactivate living organism. It is done by heating,
irradiation and treatment with certain chemicals.
(iv) Inoculation - Artificial introduction of microorganism or other substances into body or into a
culture medium is known as inoculation. Substance, containing microorganism that is introduced
in inoculation is called inoculum. The medium is inoculated with pure culture of selected micro-
organisms to achieve fermentation.
(v) Maintenance of pH and temperature - Optimum pH and temperature is very necessary for
activities of microorganism to produce desired products in sufficient quantity.
(vi) Recovery of Products - After fermentation the desired product is recovered.
 Drawbacks in Stirred bioreactor - it is relatively expensive. It is mainly due to high energy
requirements to drive agitators.
6. Downstream processing - Product obtained is subjected to a series of processes (collectively
called downstream processing) before it is made into finished product ready for marketing. The
two main processes are (a) separation (b) purification
 Product is then formulated with suitable preservatives. Such formulations have to undergo
clinical trials in case of drugs. A proper quality control testing for each product is also needed.
Molecular Probes - Molecular probes are small DNA or RNA segments that are used to detect
presence of complementary sequences in nucleic acid samples.
 Probes are made up of 200-500 nucleotide sequences. Probes are labelled with radioactive or
non-radioactive compound.
Types of Probes
1. cDNA Probes – DNA probe formed by reverse transcription of mRNA is called cDNA.
2. Genomic DNA Probes - These are double stranded DNA molecules. They are either specific
probe for identification of homologous sequences in genome while random probes are used for
study of DNA polymorphism and construction of molecular maps.
3. Single Stranded DNA Segments - Single stranded DNA segments are best type of probes.
These are prepared by cloning concerned DNA in a vector like phage M-13 vector of E. coli.
4. Synthetic Oligo-nucleotides - Synthetic probes with known nucleotide sequences can be
synthesized chemically by automated DNA synthesizers. These oligo-nucleotide probes prove to
be efficient only when they range between 20-40 nucleotides long.
 Probes do not transmit specific signals by which their presence can detected. They are labelled
either with radioactive isotopes or with non-radioactive signal molecules.
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Applications of Probes
1. Accurate diagnosis of pathogenic diseases.
2. Preparation of genome maps of plants, animals, viruses and bacteria.
3. Development of restriction fragment length polymorphism (RFLP) maps.
4. Isolation of gene or related sequences.
5. Identification of recombinant clone carrying desired DNA insert.
Molecular Marker - Molecular marker is a DNA fragment that is associated with certain trait(s).
Markers help in determine the location of genes that control important traits. Molecular marker is
of two types - (a) anonymous (b) defined
 Anonymous marker is cloned random DNA fragment whose function or specific features are
not known. Defined marker contains gene or some other specific feature.
Selectable marker – If ampicillin resistant gene is transfer in to E. coli. Now E. coli become
resistant to ampicillin. If transformed cells are spread on agar plates having ampicillin, only
transfermants grow but untransformed recipient cells die. Hence, due to ampicillin resistance
gene, one is selected as transformed cell in presence of ampicillin. Ampicillin resistance gene is
called as “selectable marker”.
Types of Molecular Markers
A. Southern Hybridization Based markers —Restriction fragment length polymorphism (RFLP)
B. Polymerase chain reaction (PCR) based markers
 Randomly amplified polymorphic DNAs (RAPDs)
 Variable number of tandem repeat (VNTR)
VNTR (Variable Number of Tandem Repeats) - VNTR are molecular makers also called
polymorphic markers because they have different structures in different individuals of a
species. VNTR are dispersed throughout genome and are made up of a variable number of end-
to-end duplications of identical sequences of 2-80 bp each.
Types of VNTR - 1. Minisatellite DNAs 2. Microsatellite DNAs
 These together constitute hyper-variable region or DNA.
(i) Mini-satellite DNAs are 200 to 2000 bp long and consist of tandem repeats of 15 to 60 bp
sequences. In humans, minisatellite DNAs are concentrated in proterminal regions of
chromosomes, hence, they do not constitute a good marker system for human genome.
(ii) Micro-satellite DNAs are short sequences of less than 100 bp and are made up of tandem
repeats of 2 to 7 bp, hence, they are also called short tandem repeats (STRs), simple sequence
repeats (SSR) and simple sequence length polymorphism (SSLP).
Measurement of DNA - DNA in a cell is measured in terms of DNA content in 1C (G1 phase of
cell cycle) cells or genome size in a haploid cell.
 DNA content in a 1C human cells is 3.2 picograms.
 DNA size in a haploid human cell is 3.2 x l09 bp.
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Foreign DNA (Passenger DNA) - DNA which is transferred from one organism into another by
joining it with vehicle DNA, is called foreign DNA. Three types of passenger DNAs are used.
1. Complementary DNA (cDNA) 2. Synthetic DNA (sDNA) 3. Random DNA
1. Complementary DNA (cDNA) - It is formed on RNA
template with help of RNA dependent DNA polymerase
called reverse transcriptase enzyme.
 DNA is separated from RNA-DNA complex in presence of
alkaline phosphatase enzyme. A cDNA strand is formed on
separated single-stranded DNA template with help of DNA
polymerase enzyme.
2. Synthetic DNA (sDNA) - It is synthesized on DNA
template or without a template.
 Artificial Synthesis of DNA on Template – Kornberg produced DNA from nucleotide in
presence of DNA polymerase enzyme and a segment of viral DNA which acts as a primer.
 Artificial Synthesis of DNA without a Template - Hargobind Khorana synthesized gene
coding for tyrosine-tRNA of E. coli. The gene had 207 nucleotide pairs.
3. Random DNA - Small fragments are formed by breaking a chromosome of an organism in
presence of restriction enclonucleases.
Difference Between rDNA and cDNA
rDNA cDNA
1. It is the DNA formed by joining together DNA 1. It is DNA obtained from an RNA template
from two different organisms. using enzyme reverse transcriptase.
Differences Between Plasmid DNA and Chromosomal DNA
Plasmid DNA Chromosoma1 DNA
1. It is always double stranded. 1. It may be single stranded or double
stranded.
2. It is circular. 2. It is linear or circular.
3. It is naked without histone protein. 3. It is coated with histone protein.
4. It does not carry any vital gene necessary for 4. It carries vital genes necessary for cell.
cell.
5. It can replicate independent of main genoine. 5. It replicates with genome.
6. It does not act as genetic factor. 6. It acts as genetic factor.
7. Introns are absent. 7. Both exons and introns are present.
 Chimaeric Gene - In recombinant DNA technology, a gene constructed by combining coding sequence from one
gene with regulatory sequences of another gene (usually of a different organism) is called chimaeric gene.
 Chimaeric DNA - Chimaeric DNA molecules are produced by inserting a foreign DNA sement into the DNA
molecule of a vector.
 Molar concentration is the ratio of the number of moles of solute in a solution divided by the volume of the
solution expressed in litters.
 Dextran is a plasma extender and is used in blood transfusions.
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 New weeds, insects pests and diseases could also come into our country alongwith the introduced vaneties.
Argemone mexicana is an example of a weed that en tere India with an introduction. Therefore; all introductions are
carefully examined for the presence of weeds, insects and disease- causing organisms; this is known as quarantine.
 Antony van Leeuwenhoek discovered bacteria.
 Humans have produced a new allopolyploid crop called triticale in the following manner Aflotetraploid wheat
(Triticum turgidum) was hybridised with rye (Secale cereale; a diploid species). The chromo som number of the
resulting F1 was doubled to produce triticale. Triticabe is cultivated in some areas of Punjab and in the hilly regions
of the country.
 DNA is bigger than enzyme.
 Ti Plasmid - Agro bacterium tumefaciens contains a large plasmid which induces tumour in plants, therefore, the
plasmid is called as Ti plasmid. The size of Ti-plasmid ranges between 180-250 Kb.
 Most sensitive technique to detect malignant cell in Non-Hodgkin’s lymphoma is PCR Polymerase Chain Reaction).
 Gene splicing or Isolation of DNA. Following are some techniques
to isolate or synthesise the gene or the DNA fragment.
(i) Fragmentation of DNA by cleaving with restriction enzymes.
(ii) Artificial synthesis of gene.
(iii) Synthesis of complementary DNA (cDNA).
 Most popular eukaryotic host organism is yeast specially Saccharomyces cerevisiae (baker’s yeast). Transforming
yeast cells is relatively easy, as they possess the machinery necessary for the modification of proteins. On the other
hand, it is sometimes necessary to clone a gene in a more specific type of eukaryotic cell, for example, in a
mammalian cell or plant cell.
Important Recombinant Proteins and Their Therapeutic Uses
Recombinant Proteins Therapeutic Uses
1. OKT-3 Used for reversal of acute kidney transplantation rejection.
OKT-3 is therapeutic antibody.
2. ReoPro Prevention of blood clots.
3. Tissue Plasminogen activator (t-PA) Used for acute myocardial infarction.
4. Asparaginase Treatment of some types of cancer.
5. DNase Treatment of cystic firbrosis
6. Human Insulin (Hurnulin) Treatment of diabetes mellitus
7. Blood Clotting Factor VIII Treatment of Haemophilia A
8. Blood Clotting Factor IX Treatment of Haemophilia B
9. Hepatitis B Vaccine Prevention of Hepatitis B
10. Platelet derived growth factor healing Approved for diabetic/skin ulcers. It also stimulates wound
11 Interferon alpha (INF -alpha) Used as vaccine for Hepatitis C
12. Hirudin Used as an anticoagulant
Brief History and Development of Genetic Engineering

(1) 1915- 1958s; Fredderick W. Twort discovered bacteriophages in 1915.
 Sharpy-Shafer (1916) first expressed that diabetes is caused by failure of the islets of pancreas to secrete a
substance named insulin.
 Term biotechnology was coined in 1917 by a Hungarian Engineer, Karl Ereky to describe a process for large scale
production in pigs.
 In 1921, Banting and Best succeeded in preparing a pure extract of insulin from the pancreatic islets of a dog with
the help of Macleod. Banting and Macleod won Nobel Prize in Medicine or physiology in 1923.
 Alexander Fleming discovered lysozyme (1922) and penicillin.
 The enzyme diastase was identified for the first time in 1933 by Paven and Persoz.
 Gel electrophoresis technique was developed by A. Tiselius in 1937. He got Nobel Prize in chemistry in 1948.
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 Plasmids were discovered by Willam Hays and Joshua Lederberg in 1952.
 Zinder and Lederberg discovered transduction in 1952. Transduction is transfer of DNA from one organism to
another through a bacteriophage.
 Sanger worked out the molecular structure of insulin in 1954.
 The enzyme DNA Polymerase was investigated by A. Kornberg in E. coli in 1956.
(2) 1960s-1970s; Isolation of restriction enzymes and their use to analyse DNA structure. The presence of restriction
endonuclease was postulated by W. Arber during 1960s.
 Tsan synthesized human insulin first time in 1965. Molecular basis of Restriction Modification System was
explained first by Werner Arber in 1965.
 In 1968, Smith discovered the first restriction endonuclease Hind II from Haemophiius influenzae.
 In 1970, Haemilton Smith et al, isolated first ‘restriction endonuclease’ from bacterium Haemophilus
parainfluenza.
(3) 1971-1975; DNA cloning techniques involving recombinant DNA developed. First gene cloned (bacterial). In 1971,
Merill, Geier and Petricciani reported the transfer of a gene from bacterium Escherichia coli into cultured human
cells through an intermediate virus.
 In 1972, Berg was able to introduce a gene of SV-40 into a bacterium with the help of lambda phase. In 1972;
Genetic engineering was started by Paul Berg, Herbert Boyer, Annie Chang and Stanley Cohen, who generated first
recombinant DNA molecule by combining a gene from a bacterium with the plasmid of E. coli. Paul Berg-Father of
genetic engineering, transferred gene of SV-40 Virus into E. coli.
 1974; First expression in a bacterium of a gene from a different species. The National Institute of Health (NIH),
USA established the Recombinant Advisory Committee (RAC). In 1975; Milstein and Kohier developed hybridoma
technology for the production of monoclonal antibodies.
(4) 1977; Boliver and Rodriguez considered the first artificial cloning vector pBR 322. First complete genetic code of
an organism and its generic code is 5375 bases long.
(5) 1978; W. Arber, H. Smith and D. Nathans got Nobel prize in medicine or physiology for discovery of restriction
endonucleases. Bacteria produced human somatostatin from a synthetic gene. Later the same year bacteria also
produced human insulin.
(6) 1979; Collins and Hohn developed a method for obtaining recombinant plasmids packed into the protective coats of
phage and then transduced into E. coli.
(7) 1980; Berg along with Walter Gilbert and Frederick Sanger got Nobel Prize in Chemistry.
(8) 1981/82; First transgenic animals (mice) produced.
(9) 1982; Insulin (Eli Lilly’s Humulin) is the first product made by genetically engineered bacteria to be approved for
use in Britain and the USA.
(10) 1983; First transgenic plants produced. Eli Lilly, an American company has started selling Humulin, the
commercially produced human insulin since 5th July, 1983.
(11) 1985; First transgenic farm animals produced (rabbits, pigs and sheep). Kary Mullis invented the Polymerase Chain
Reaction or PCR. Kary Mullis shared the Nobel Prize with Michael Smith in chemistry in 1993.
(12) 1986; First controlled release of genetically engineered organisms into the environment.
(13) 1989; First patented transgenic animal, the onco-mouse.
(14) 1990; Human genome project started. First successful gene therapy for SCID in USA.
(15) 1990-1992; First transgenic cereal plants (Maize and Wheat).
(16) 1992; Regulations for deliberate release of genetically engineered organisms established in the USA and EU.
(17) 1993; First human gene therapy trial in UK. Gene therapy for cystic fibrosis and SCID begun in UK.
(18) 1994; Genetically engineered tomato marketed in the USA.
(19) 1996; Genetically engineered tomato marketed in Britain.
(20) 1997; First cloned mammal produced from a single cell. The sheep, Dolly was developed from a single udder cell.
(21) In 1998; Eli Lilly and Ranbaxy launched diabetic drugs like Humapen, Humalog and protein Kinase C.
(22) Human Genome Project was started in Oct., 1990 and completed in April, 2003. The human genome consists of
about 30,000 genes.
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BIOTECHNOLOGY AND ITS APPLICATION
 Biotechnology deals with industrial scale production of biopharmaceuticals and biological using
genetically modified microbes, fungi, plants and animals.
 Applications of biotechnology include therapeutics, diagnostics and genetically modified crops
for agriculture, processed food, bioremediation, waste treatment and energy production.
Research Areas of Biotechnology
1. Providing best catalyst in form of improved organism like microbe or pure enzyme.
2. Form optimal conditions by engineering for a catalyst to act.
3. Processing technologies to purify protein/organic compound.
Biotechnological Applications in Agriculture
(a) Agro-chemical based agriculture
(b) Organic agriculture
(c) Genetically engineered crop-based agriculture
 Green Revolution increase food production but it was not sufficient to feed growing human
population. Increased yield is due to use of improved crop varieties, its reasons are better
management practices and use of agrochemicals such as fertilizers and pesticides. But
agrochemicals are expensive for farmers of developing countries.
 One of reasons to decrease the yield is use of conventional breeding processes.
 One solution of this problem is use of genetically modified crops.
Genetically Modified Organisms (GMO) – when genes of plants, bacteria, fungi and animals have
been manipulated, such organisms are called Genetically Modified Organisms.
 Behaviour of a GMO depends on the nature of genes transferred, nature of host plants, bacterium
and animal. Food web also plays important role.
Transgenic Plants - Plants produced through genetic engineering contain gene of usual unrelated
organism, such genes are called transgenes and plants having transgenes are called transgenic
plants.
 Recombinant DNA techniques are being used to improve crop plants by increasing their
activity by making them more nutritious and disease resistant. Transgenic plants have a natural
resistance to herbicides and pests.
Application of DNA Technology in production of Transgenic Plants
Insect Resistance plant
 Bt-Cotton - Soil bacterium Bacillus thuringiensis
produces proteins that kill insects like Lepidopterans
(tobacco budworm, armyworm), coleopterans
(beetles) and dipterans (flies, mosquitoes). B.
thuringiensis forms some protein crystals. These
crystals contain toxic insecticidal protein.
 This toxin not kill Bacterium because – In bacterium
Bt-toxin proteins present in inactive protoxins, when
insect ingests this inactive protein it converted into
active toxic form due to alkaline pH in alimentary
canal.
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 This activated toxin binds to surface of midgut epithelial cells and create pores, which cause cell
swelling, lysis and finally cause death of insect.
 This Bt-toxin genes were isolated from Bacillus thuringiensis and incorporated into several crop
plants like cotton. Choice of genes depends upon crop and targeted pest.
 Toxin is coded by a gene named cry. Cry genes
GENE PLANT INSECT
cry I AC and cry II Ab have been incorporated in cry I Ab Corn Corn borer
cotton. This genetically modified crop is called cry II Ab Cotton Cotton bollworms
Bt-cotton because it contains Bt-toxin genes cry I Ac Cotton Cotton bollworms
against cotton bollworms.
 Similarly, cry I Ab has been introduced in Bt-corn to protect from corn borer.
Ti plasmid - Agrobacterium tumefaciens a soil bacterium use as vector to transmit gene in to
plant by Ti-plasmid (Tumor inducing plasmid). This plasmid induces tumors in broad leaf plants
such as tomato, tobacco and soybean. Ti-plasmid use as vector; researchers eliminated its tumor
causing properties while keeping its ability to transfer DNA into plant cells. This bacterium is
called natural genetic engineer because genes carried by this plasmid produce effect in several
parts of plant.
1. Agrobacterium infects all broad-leaved agricultural crops like tomato, soybean, sunflower and
cotton. It does not infect cereals. It induces formation of cancerous growth crown gall tumor.
This tumor formation is due to Ti-plasmid. By genetic engineering tumor formation character of
gene deleted. But it still infects plants.
 Agrobacterium infect plants cells and cause tumor, tumored cell secretes substance on which this
bacterium feed. By genetic engineering this bacterium Ti-plasmid modified and their pathogenic
nature remove but this Ti-plasmid still infecting plant cells.
2. Ti-plasmid, which transferred into plant cell DNA, is called T-DNA. This T-DNA with desired
DNA is inserted into chromosomes of host plant where it produces copies. This plant transferred
into soil and expresses them self as new plant which contain new genes.
Pest Resistance Plant
 RNA interference (RNAi) - Many
nematodes infect plants and animals. A
nematode Meloidogyne incognitia
infects roots of tobacco plants and
reduce its yield.
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 To reduce this infection a strategy was adopted, which is based on process of RNA interference
(RNAi). RNAi is the phenomenon of inhibiting activity of a gene through production of sense
and antisense strand.
 RNAi takes place in all eukaryotes organisms as a method of cellular defense. This method
involves a specific mRNA silencing. It is due to a complementary dsRNA molecule which binds
to and prevents translation of mRNA (silencing).
 Source of this dsRNA from an infection by viruses having RNA genomes or mobile genetic
elements (transposons), which replicate through an RNA intermediate.
 Nematode specific genes introduced into host plant, by using Agrobacterium vectors.
Introduction of DNA was such that, it produces both sense and anti-sense RNA in host plant.
These two RNAs being complementary to each other formed dsRNA that initiated RNAi and
silenced specific mRNA of nematode. Result of that the parasite could not survive in transgenic
host due to interfering RNA. Hence, transgenic plant got itself protected from parasite.
Produce a protein of interest-Hirudin
 It produces a protein that is the product in which we are interested. Hirudin protein present in
leech which prevents blood clotting. Its gene was introduced in Brassica napus. The seeds of
Brassica napus start forming hirudin which extract and purified.
Flavr Savr’ Tomato - A GM plant – (Maintain Post harvesting loss)

 Enzyme poly-galacto-uronase is responsible for break downing
of cell wall of ripening tomato. If this enzyme becomes non-
active by inactivation of gene, the fruit remains fresh for long. It
retains flavour, contain superior taste and higher quantity of total
soluble solids.
Golden Rice
 It is a transgenic variety of rice (Oryza sativa) which contains
good quantities of β-carotene (provitamin A). β-carotene is a
principal source of vitamin A. Grains of rice are yellow in colour
due to β-carotene, this rice is commonly called golden rice.
Prof. Ingo Potrykus and Peter Beyer produced GM rice by
introducing three genes associated with synthesis of carotene.
Golden Rice- A japonica variety of rice was engineered with three genes necessary for rice grain to produce and store β-carotene.
These included two genes-daffodil plant and third from bacterium. Researchers used a plant microbe to ferry in genes into plant
cells.
Genetically Modified Crops - They contain one or more useful foreign genes or transgenes.
Applications of GM Plants
1. GM crops are pest resistance; these GM crops help to reduce use of chemical pesticides.
2. GM crops have more tolerant to abiotic stresses (cold, drought, salt, heat, etc.)
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3. They have helped to reduce post harvest losses.
4. Increased efficiency of mineral usage by plants prevents early exhaustion of fertility of soil.
5. GM plants enhance nutritional value of food, e.g., vitamin-A enriched rice.
6. Herbicides (weed killers) do not harm the GM-crops.
7. GM-plants show great tolerance against disease.
8. Phyto-remediation - Plants like poplar trees have been genetically modified to clean heavy
metal pollution from contaminated soil.
9. CPTI (Cow-Pea Trypsin Inhibitor) gene has been introduced in tobacco to show resistance
against pests.
Criticism against GM crops
1. Can harm to other organisms - A laboratory study by ‘Nature’ state that pollen of Bt-corn
caused high mortality in monarch butterfly caterpillars. Monarch caterpillars feed on milkweed
plants, not on corn, but fear is that if pollen from Bt-corn is reach to other milkweed plants by
wind in neighbour fields and caterpillars eat the pollen and perish. Although the ‘Nature’ study
was not conducted under natural field conditions, results seemed to support this viewpoint.
2. Reduced effectiveness of pesticides - Like populations of mosquitoes become resistance to
pesticide DDT, some people says that insects also become resistant to Bt or other crops that are
genetically modified to their own pesticides.
3. Gene transfer to non-target species - Another concern is that GM crop plants crossed with
weeds and become resistant and become “super weeds”.
Human health risks –
A. Transgenic food may cause toxicity or produce allergies because it is a foreign protein.
B. Bacteria present in human alimentary canal can take up antibiotic resistance gene that is present
in GM food and become resistant to concerned antibiotic and difficult to manage.
C. Economic concerns. To carry a GM food in market is a lengthy and costly process and agri-
biotech companies want profitable return.
Transgenic Animals - Animals which carry foreign genes are called transgenic animals.
Advantages of Transgenic Animals
 Transgenic animals are use in formation of human protein like - α-1-antitrypsin, which used to
treat emphysema. Similar attempts are being made for treatment of phenylketonuria (PKU) and
cystic fibrosis.
 In 1997, first transgenic cow, Rosie, produced human protein- enriched milk (2.4 gms per
litre). This milk contained human alpha-lacta-albumin. It is a more balanced product for
human babies than natural cow-milk.
 Transgenic mice are use for testing for safety of vaccines before they use on human. Transgenic
mice are being used to test the safety of polio vaccine.
 Transgenic animals (pig) are use to develop spare parts like heart,
pancreas for human.
Transgenic Fish (Transgenic Salmon) – GM salmon was the first
transgenic animal for food production. Genetically modified
sperms were fused with normal ova (eggs) of same species.
Zygotes which developed into embryos gave rise to much bigger
adults than parent. Transgenic salmon possesses an additional gene
that codes for growth hormone that allows fish to grow larger more
than non-transgenic salmon.
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Transgenic Chicken - Avain leukosis virus (ALV) is a serious viral pathogen of chickens.
Salter and Crittenden (1988) have produced an ALV-resistant strain of chicken by introducing
a defective genome of this virus into genome of chicken. This principle is also applied to evolve
transgenic fish that can resist viral infections.
Transgenic Mice - Mouse is most preferred mammal for studies
on gene transfers due to short oestrous cycle and gestation period,
relatively short generation time, production of several offspring per
pregnancy, convenient in vitro fertilization, successful culture of
embryos in vitro, etc. As a result, the techniques for gene transfer
and transgenic production have been developed using mice as
models in other animals. Recently, rats and rabbits are being used for
research work on gene transfer.
Transgenic Dogs - Dogie is a transgenic dog with excellent smelling power. It was used during
attack on World Trade Centre (WTC) of USA in 2001 to recover injured people from heaps of
devastated building.
ANDI - DNA of jelly fish was introduced into unfertilized egg of a Rhesus monkey in test tube.
The diploid egg went cleavage and early embryo was implanted in a surrogate mother. ANDI,
the first transgenic monkey was born on Oct 2, 2000. This work would be helpful for curing
diseases like as breast cancer, Alzheimer’s desease, diabetes and AIDS.
Biotechnological Applications in Medicine
 About 30 recombinant therapeutics have been approved for human use in world over. In India,
12 of these are presently being marketed.
Genetically Engineered Insulin
 Sharpy-Shafer (1916) first expressed that diabetes is caused by failure of islets of pancreas to
secrete a substance named by him as insulin. Insulin is secreted by the Beta cells of islets of
Langerhans of pancreas. In 1921, Banting and Best succeeded in preparing a pure extract of
insulin from pancreatic islets of a dog with Macleod. Banting and Macleod won the 1923 Nobel
Prize in Medicine or Physiology.
 They demonstrated that administration of insulin could cure diabetes in human beings. Later,
insulin for curing diabetes used to be extracted from pancreas of slaughtered pigs and cattle. This
insulin is slightly different from human insulin and brings about some undesirable side effects
such as allergy.
25
 Human insulin is made up of 51 amino acids arranged in two polypeptide chains. A having 21
amino acids and B with 30 amino acids. Two polypeptide chains are interconnected by two
disulphide bridges or S-S linkages.
 One S-S linkage also occurs in A chain.
 Pro-insulin has three chains, A, B and C. C-chain has 33 amino acids, this chain remove to form
active insulin.
 Bacteria can not be made to synthesize insulin from its gene because of the presence of introns.
Bacteria do not possess enzymes for removing intron mediated transcription.
 In mammals, including humans, insulin is
synthesized as a pro-hormone, which
contains an extra C-peptide. This C-peptide
is not present in the mature insulin and is
removed during maturation.
 The main challenge for production of insulin
using rDNA technique was getting insulin
assembled into a mature form.
 In 1983, Eli Lilly an American company,
first prepared two DNA sequences
corresponding to A and B chains of human
insulin and introduced in plasmids of E. coli.
Chains A and B were produced separately
and combined by creating disulfide bonds to
form human insulin (humulin).
 Molecular structure of insulin worked out
by Sanger. Tsan synthesized human insulin
first time. Insulin is not administered orally.
Gene Therapy
 Gene therapy is a collection of methods that allows correction of a gene defect or Gene therapy
is technique of genetic engineering in which ‘a faulty gene’ replaces by a normal healthy
functional gene. Gene therapy is being tried for sickle cell anaemia and severe combined
immuno-deficiency (SCID).
Adenosine De-Aminase (ADA) Deficiency
 First clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase
(ADA) deficiency. This enzyme is very important for immune system for function. SCID is
caused due to defect in gene which synthesized enzyme adenosine deaminase.
 In some children ADA deficiency can be cured by bone marrow transplantation.
 In others it can be treated by enzyme replacement therapy, in which functional ADA is given to
the patient by injection. But in both approaches patients are not completely cured. Because these
patients do not have functional T-lymphocytes, they cannot provide immune responses against
invading pathogens.
 By gene therapy, lymphocytes, a kind of white blood cells, are extracted from the bone marrow
of patient and are grown in a culture outside the body.
26
 A functional ADA gene or
cDNA (using a retroviral
vector) is then introduced
into lymphocytes and
lymphocytes are re-injected
to patient’s bone marrow.
But these cells do not
always remain alive; the
patient requires periodic
infusion of such genetically
engineered lymphocytes.
 However, if the isolated
gene from bone marrow
cells producing ADA is
introduced into cells at
early embryonic stages, it
can be a permanent cure.
Molecular diagnosis
 Recombinant DNA molecules and techniques like PCR (Polymerase Chain Reaction) are used
for early diagnosis of disorders.
 Cloned genes when expressed to produce recombinant proteins help in developing sensitive
diagnostic techniques like ELISA. The cloned genes are also used as probes’ to detect the
presence of complementary DNA strand.
 A probe is a piece of single stranded DNA, that is tagged with a radioactive molecule and it is
used to find us complementary DNA by hybridization.
 It is followed by detection of radioactivity by autoradiography. Presence of a normal or mutant
gene can be detected using such a method. PCR is used to detect HIV and to detect mutations in
gene.
Ethical Issues
 Genetic modification of organisms can have unpredictable/undesirable effects when such
organisms are introduced into the ecosystem. The modification and use of such organisms for
public services has also resulted in problems with the granting of patents.
 Hence, the Indian Government has set up organizations which are authorized to make decisions
regarding the validity of genetic modifications and the safety of introducing genetically modified
organisms for public services. One such organization is the Genetic Engineering Approval
Committee (GEAC).
Bio-piracy
 Biopiracy refers to use of bio-resources by multinational companies and other organizations
without proper authorization from the countries and people concerned.
 Industrialized/developed nations are rich financially, but poor in biodiversity and traditional
knowledge, while the developing and underdeveloped countries are rich in bio-resources and
traditional knowledge.
27
 Some such developed countries use the bio-resources and traditional knowledge of the other
countries without proper authorization and\or compensation to counties concerned (fliopiracy).
 Basmati rice grown in India is distinct for its unique flavour and aroma, hut an American
company got patent rights on Basmati through the US percent and trademark office; the new
variety of Basmati has been developed by this company by crossing an Indian variety with the
semi-dwarf varieties.
 Now some nations are developing laws to prevent such unauthorized exploitation of their bio-
resources and traditional knowledge.
********
 Trichoderma harzianum is a fungus that is also used as a fungicide. It is used
for foliar application, seed treatment and soil treatment for suppression of various disease
causing fungal pathogens.
Very short answer type questions
Q.1 Why is the gene encoding for Cry’ protein inserted into a crop plant?
Q.2 Crystals of Bt toxin produced by some bacteria do not kill the bacteria themselves because
(a) bacteria are, resistant to the toxin (b)toxin is immature
(c) toxin is inactive. (d) bacteria enclose toxin in a special sac.
Q.3 Does our blood have proteases and nucleases?
Q.4 Define biotechnology.
Q.5 Write the scientific name of the nematode that attacks the roots of tobacco plants.
Q.6 Name the bacterium that is used as a vector to insert genes in crop plants.
Q.7 What are probes/
Q.8 What are transgenic animals?
Q.9 Name two diseases that can be treated by producing biological compounds in transgenic animals.
Q.10 Name the first transgenic cow.
Q.11 Which vaccine was being tested on mice
Q.12 Define biopiracy.
Q.13 What is meant by ‘traditional knowledge?
Q.14 Expand GEAC.
Short answer type questions
Q.1 What arc geneticafly modified organisms ? Name two factors on which their behaviour depends.
Q.2 What are the advantages of molecular diagnostics over conventional methods ?
Q.3 Mention two uses of cloned genes in molecular diagnostics.
Q.4 How is early detection of infectious diseases possible by molecular diagnosis?
Q.5 Give two reasons why Indian Government has set up organisadons to monitor GM research and introduction of
OMO for public services?
Q.6 Give the scientific name of the soil bactenum which produces crystal (Cry) proteins?
Q.7 How are these proteins useful in agriculture?
Q. 8 What do the differently written terms Cry’ and cry represent respectively?
Short answer type questions
3.1 Describe how nematode resistant transgenic plants have been produced.
3.2 What are transgenic bacteria ? Illustrate using any one example.
What are Cry proteins ? Name an organism that produce it. How has man exploited this protein to his
benefit?
3.4 Mention any six fields of applications of biotechnology for human welfare.
3.5 What are the three critical research areas of biotechnology
3.6 Enlist with examples the different groups of insects to which the crystal proteins are toxic.
3.8 Mention the advantages of recombinant therapeutics. How many of them have been approved world
over for human use and how many are marketed in India?
28
3.9 (i) What are the disadvantages of human use of insulin from other animal sources
3.10 Expand PCR. What is it used for in diagnostics’. What is the advantage?
3.11 Name any four transgenic animals commonly produced. Which animal constitutes a major proportion among
transgenic animals? What percentage
3.12 Write an account on the production of human insulin in transgenic organisms,
3.13 Name the biological product made in transgenic animal to treat emphysema. Explain the same.
3.14 How many documented varieties of Basmati rice are grown in India? How has this variety of rice
been exploited?
Long answer type questions
5.1 Compare and contrast the advantages and disadvantages of production of genetically modified crops.
5.2 What is gene therapy ?fllustrate using the example of adenosine deaminase (ADA) deficiency.
5.3 Diagrammatically represent the experimental steps in cloning and expfessing a human gene (say the
gene for growth hormone), into a bacterium like E. co/i. have helped to meet the increased demand
for food.
5.4 (i) What are the three options that can be thought of to increase food production?
(ii) Mention the reasons for the success of Green Revolution in increasing food production.
(iii) Why do we still search for other alternatives to increase food production?
5.5 Describe the different ways in which genetic modification or genetically modified plants (U) What
are the concerns about transgenic insulin?
5.6 Name the soil bacterium that produces a protein/chemical that is toxic to insect pests. Show with
example that the different forms of them encoded by different forms of the gene are insect specific.
5.7 Describe giving example for each, why transgenic animals are produced.
I
29
Exercise–1
Section–A
Q.1 Which of the following can't be related to the Q.10 During gel electrophoresis for separation of DNA
biotechnology? fragment
(1) Integration of natural science and organisms (1) Smallest fragment will move to the farthest point
(2) Techniques to alter the chemistry of DNA towards cathode
(3) Introducing undesirable genes into the target (2) Smallest fragment will move to the farthest point
organism towards anode
(4) Maintenance of sterile ambience to enable growth of (3) Largest fragment will move to the farthest point
only the desired microbes towards cathode
Q.2 Which of the following specific DNA sequence is (4) Largest fragment will move to the farthest point
responsible for initiating replication? towards anode
(1) Vector site Q.11 After electrophoresis, the separated DNA
(2) Restriction enzymes action site fragment can be visualised in ethidium bromide gel
(3) 'Ori' site exposed to UV light. These DNA fragments appear as
(4) Palindromic site ___ coloured bands
Q.3Autonomously replicating circular extra (1) Orange (2) Blue
chromosomal DNA of prokaryotic cell is called (3) Silver (4) Green
(1) Satellite DNA (2) Plasmid Q.12 The procedure through which a piece of DNA is
(3) Recombinant DNA (4) Nucleoid introduced in a host bacterium is called
Q.4 Key tools to be involved in recombinant DNA (1) Cloning (2) Transformation
technology are (3) PCR (4) Clonal selection
A. Restriction enzymes B. Polymerase enzyme Q.13 After completing the transformation experiment
C. Ligase enzymes D. Vectors involving the coding sequence of enzyme -
(1) A only (2) A & C only galactosidase, the recombinant colonies should
(3) A, B & C (4) A, B, C & D (1) Give blue colour (2) Not give blue colour
Q.5 First restriction endonuclease to be discovered was (3) Have active-galactosidase (4) Both (2) & (3)
(1) Hind II (2) Eco RI Q.14 Which of the following has the ability to
(3) Bam HI (4) Pst I transform normal cells into cancerous cells in animals?
Q.6 Approximately how many restriction enzymes have (1) Agrobacterium tumifaciens (2) Retroviruses
been isolated from the different (over 230) strains of (3) DNA –viruses (4) Plasmids
bacteria? Q.15 Which of the following is not applicable to
(1) 300 (2) 600 Agrobacterium tumifaciens?
(3) 750 (4) 900 (1) Pathogen of several dicot plants
Q.7 The conventional method for naming the restriction (2) Has ability to transform normal plant cells
enzymes is followed. In case of Eco RI, the 'R' indicates (3) Delivers gene of our interest
(1) Genus (2) Species (4) Ti plasmid of it is always pathogenic to plants
(3) Name of the scientist (4) Strain without any exception
Q.8 The restriction endonuclease enzyme binds to the Q.16 Insertional inactivation is related to
DNA & cut (1) Microinjection (2) Gene gun
(1) Anyone strand of the double helix (3) Gel electrophoresis (4) Selection of recombinants
(2) Each of the two strands at specific points in their Q.17 For transformation with recombinant DNA, the
base - sugar bonds bacterial cells must first be made 'competent' which
(3) Each of the two strands at specific points in their means
base - phosphate bonds (1) Should increase their metabolic reactions
(4) Each of the two strands at specific points in their (2) Should decrease their metabolic reactions
sugar - phosphate backbones (3) Increase efficiency with which DNA enters
Q.9 Which of the following palindromic sequence is bacterium
recognised by Eco RI? (4) Ability to divide fast
Q.18 Which of the following method can be used for
making the bacterial cell 'competent'?
(1) Treating with specific concentration of divalent
(1) (2) cation (Ca2+)
(2) Treating with specific concentration of monovalent
cation (K+)
(3) Heat shock
(3) (4) (4) Both (1) & (3)
Q.19 Which of the following techniques can be used to
introduce foreign DNA into cell?
30
(1) Using disarmed pathogen (2) Microinjection (3) If the alien piece of DNA is linked with origin of
(3) Gene gun 4) All of these replication in chromosome it will replicate
Q.20 During heat shock to the bacterium, the (4) All of these
temperature used for giving thermal shock is Q.29 In the year 1963, the two enzymes responsible for
(1) 82°C (2) 100ºC restricting the growth of bacteriophage in Escherichia
(3) Liquid nitrogen (4) 42ºC coli were isolated. They were ___ and ___ respectively
Q.21 Which of the following enzyme is used in case of (1) Ligase, Restriction endonuclease
fungus to cause release of DNA along with other (2) Helicase, Restriction endonuclease
macromolecules? (3) Methylase, Restriction endonuclease
(1) Lysozyme (2) Cellulase (4) DNA polymerase, Restriction endonuclease
(3) Chitinase (4) Amylase Q.30 The cutting of DNA by restriction endonucleases
Q.22 During isolation of DNA, addition of which of the results in the fragments of DNA. These fragments are
following causes precipitation of purified DNA? generally separated by a technique known as
(1) Chilled ethanol (2) Ribonuclease enzyme (1) Gel-filtration chromatography
(3) DNA polymerase (4) Proteases (2) Centrifugation
Q.23 Which of the following is the correct sequence of (3) Gel electrophoresis
PCR or polymerase chain reaction? (4) Thin layer chromatography
(1) Denaturation ↔ Annealing ↔Extension Q.31 Which of the following bacteria are known as
(2) Extension ↔Denaturation ↔Annealing natural genetic engineers of plants' as gene transfer is
(3) Annealing ↔Extension ↔Denaturation happening in nature without human interference?
(4) Denaturation↔ Extension↔ Annealing (1) Azotobacter (2) Agrobacterium tumefaciens
Q.24 The most commonly used bioreactor is of stirring (3) Escherichia Coli (4) Rhizobium
type. The stirrer facilitates Q.32 The technique in which a foreign DNA is
(1) Temperature control (2) pH control precipitated on the surface of the tungsten or gold
(3) Oxygen availability (4) Product removal particles and shot into the target cells is known as
Q.25 After completion of biosynthetic stage , the (1) Microinjection
separation and purification of product is called (2) Chemical-mediated genetic transformation
(1) Upstream processing (2) Downstream processing (3) Electroporation
(3) Modern biotechnology (4) Gene amplification f (4) Biolistics
Q.26 From isolated DNA from a cell culture with the 7 Q.33 Isolation of the genetic material in pure form, free
desired gene, DNA segment can be excised from other macromolecules can be achieved
by 'molecular scissors' or 'chemical scalpels' what by treating the bacterial cells/plant or animal tissues
biotechnologists call as ______ with following enzymes except
(1) Polymerase enzymes (2) DNA ligase (1) Lysozyme (2) Cellulase
(3) Restriction enzymes (4) Helicase (3) Chitinase (4) Ligase
Q.27 All the following statements about Stanley Cohen Q.34 Which of the following is not a recombinant
and Herbert Boyer are correct but one is protein used in medical practice?
wrong. Which one is wrong? (1) TPA (tissue plasminogen activator)
(1) They discovered recombinant DNA (r-DNA) (2) Interferon (α, β , and γ)
technology and this marks the birth ofmodern (3) Vaccine (for hepatitis B)
biotechnology· (4) Heparin
(2) They first produced, healthy sheep clone, a Finn Q.35 cDNA is
Dorset lamb, Dolly, from the differentiated adult (1) Circular DNA in the bacterias
mammary cells (2) Complemantary DNA
(3) They invented genetic engineering by combining a (3) Copy DNA
piece of foreign DNA containing a gene from a (4) Both (2) & (3)
bacterium with a bacterial plasmid using the enzyme Q.36 Noble prize of 1978 for restriction endo-nuclease
restriction endonuclease technology was given to
(4) They isolated the antibiotic resistance gene by (1) Temin and Baltimore
cutting out a piece of DNA from a plasmid which was (2) Milstein and Kohler
responsible for conferring antibiotic resistance (3) Arber, Nathans and Smith
Q.28 What is the fate of a piece of DNA, which is (4) Holley, Khorana and Nirenberg
somehow transferred into an alien organism? Q.37 Plasmids are used in genetic engineering because
(1) This piece of DNA would not be able to multiply they are
itself in the progeny cells of the organism of not (1) Easily available
integrated into the genome of the organism (2) Able to integrate with host chromosome
(2) If the alien piece of DNA had become a part of the (3) Able to replicate along with chromosomal DNA
chromosome, it will replicate (4) Contain DNA sequences coding for drug resistance
31
Q.38 Which of the following processes and techniques (2) It is often referred as 'gene splicing'
are included ·under biotechnology? (3) The organism carrying the foreign genes is termed
A. In vitro fertilization leading to a test-tube baby as transgenic or GMO
B. Synthesising gene and using it (4) Alec Jeffrey is tile father of genetic engineering
C. Developing DNA vaccine Q.44 All the following are the properties of the enzyme
D. Correcting a defective gene 'Taq polymerase' except
(1) B and D only (2) B, C and D (1) It is thermostable DNA polymerase
(3) A and B (4) A, B, C and D (2) It is isolated from a bacterium, Thermus aquaticus
Q.39 The tumor inducing (Ti) plasmid has now been (3) It is used for amplification of gene of interest using PCR
modified into a cloning vector which is no more (4) It is thermostable RNA polymerase
pathogenic to the plants but is still able to use the Q.45 Which of the following is incorrect match?
mechanisms to deliver genes of our interest into a (1) Gene therapy : An abnormal gene is replaced by
variety of plants because Ti plasmid has been modified normal gene
by (2) Cloning : Ability to multiply copies of antibiotic
(1) Adding tumor forming genes resistance gene in E. coli
(2) Deleting tumor forming genes (3) Restriction enzymes : Molecular scissors
(3) Adding genes resistant to endonucleases (4) Exonucleases : Molecular glue
(4) Deleting endonuclease Q.46 Appropriate techniques have been developed for
Q.40 Which of the following statement is incorrect? large scale cell culture using bioreactors for producing
(1) Plasmids have the ability to replicate within the (1) Foreign gene product (2) Vaccines
bacterial cells independent of the control of (3) Hormones (4) All of these
chromosomal DNA Q.47 The uptakes of genes by cells in microbes and
(2) Some plasm ids have only one or two copies per cell plants is termed as
whereas, the others may have 15-100 copies per cell (1) Insertional inactivation (2) Transformation
(3) Bacteriophages have the ability to replicate within (3) Selectable markers (4) Cloning vectors
the bacterial cell independent of the control of Q.48 If we ligate a foreign, DNA at the Bam HI site of
chromosomal DNA tetracycline resistance gene in pBR322 the recombinant
(4) Transformation is a procedure of separation and plasmid will
isolation of DNA fragments (1) Show ampicillin resistance only
Q.41 Which of the following is the first artificial (2) Show tetracycline resistance
cloning vector, which has two selectable markers, (3) Will grow well on tetracycline containing medium
tetracycline, tetR and ampR, antibiotic restriction (4) Will not grow on ampicillin containing medium
enzymes? Q.49 Polyethylene glycol can help in the uptake of
(1) YAC (2) BAC foreign DNA into the host cell, this type of gene
(3) pBR322 (4) Cosmid vectors transfer is called as
Q.42 Restriction endonucleases are the most widely (1) Electroporation
used in recombinant DNA technology. They are (2) Chemical mediated genetic transformation
obtained from (3) Microinjection
(1) Bacteriophage (2) Bacterial cells (4) Particle gun
(3) Plasmids (4) All prokaryotic cells Q.50 The normal E. coli cells carry resistance against
Q.43 All the following statements are correct about which of the following antibiotics?
genetic engineering, but one is wrong which one is (1) Ampicillin
wrong? (2) Chloramphenicol
(1) It is a technique for artificially and deliberately (3) Tetracycline or kanamycin
modifying DNA (genes) to suit human needs (4) None of these
32
Section–B
Q.1 It is theoretically possible for a gene from any ampicillin, nutrient broth plus tetracycline, nutrient
organism to function in any other organism. broth plus ampicillin and tetracycline, and nutrient
Why is this possible? broth containing no antibiotics. The bacteria containing
(1) All organisms have ribosomes the engineered plasmid would grow in
(2) All organisms have the same genetic code (1) The ampicillin and tetracycline broth only
(3) All organisms are made up of cells (2) The nutrient broth, the ampicillin broth, and the
(4) All organisms have similar nuclei tetracycline broth
Q.2 If you discovered a bacterial cell that contained no (3) The nutrient broth and the ampicillin broth only
restriction enzymes, which of the following would you (4) The nutrient broth only
except to happen? Q.6 Agrobacterium tumefaciens used in Genetic
(1) The cell would create incomplete plasmids engineering for -
(2) The cell would be unable to replicable its DNA (1) DNA-mapping (2) DNA-modification
(3) The cell would become an obligate parasite (3) Vector (4) DNA finger printing
(4) The cell would be easily infected any lysed by Q.7 A genetically engineered bacteria used for clearing
bacteriophages oil spills is -
Q.3 Assume that you are trying to insert a gene into a (1) Escherischia coli (2) Bacillus subtilis
plasmid and someone gives you a preparation of DNA (3) Agrobacterium tumifaciens (4) Pseudomonas putida
cut with restriction enzyme X. The gene you wish to Q.8 Who isolated the first restriction endonucleases-
insert has sites on both ends for cutting by restriction (1) Temin & Baltimore (2) Sanger
enzyme Y. You have a plasmid with a single site for Y, (3) Nathans & Smith (4) Paul berg
but not for X. Your strategy should be to Q.9 Genetic engineerng is -
(1) Cut the plasmid with restriction enzyme X and (1) Study of extra nuclear gene
insert the fragments cut with Y into the plasmid (2) Manipulation of genes by artificial method
(2) Cut the plasmid with restriction enzyme X and (3) Manipulation of RNA
insert the gene into the plasmid (4) Manipulation of enzymes
(3) Cut the plasmid twice with restriction enzyme Y Q.10 Which of the following enzymes cut the DNA
and ligate the two fragments into the plasmid cut with molecule at specific nucleotide sequence -
the same enzyme (1) Restriction endonuclease (2) DNA-ligase
(4) Cuticle plasmid twice with restriction enzyme Y and (3) RNA-polymerase (4) Exonuclease
ligate the two fragments onto the ends of the human Q.11 DNA finger printing was invented by -
DNA fragments cut with restriction enzyme X (1) Karl mullis (2) Alec Jeffery
Q.4 I. Transform bacteria with recombinant DNA (3) Dr. Paul Berg (4) Francis collins
molecule Q.12 Which structure involved in genetic engineering-
II. Cut the plasmid DNA using restriction enzymes (1) Plastid (2) Plasmid
III. Extract plasmid DNA from bacterial cells (3) Codon (4) None
IV. Hydrogen-bond the plasmid DNA to nonplasmid Q.13 Which of the following is the example of
DNA fragments chemical scissors -
V. Use ligase to seal plasmid DNA to non plasmid (1) ECo - RI (2) Hind-III (3) Bam - I (4) All the above
DNA. Q.14 Restriction endonucleases are used in genetic
From the list above, which of the following is the most engineering bacause -
logical sequence of steps for splicing foreign DNA into (1) They can degrade harmful proteins
a plasmid and inserting the plasmid into a bacterium? (2) They can join DNA fragments
(1) IV, V, I, II, III (2) III, II, IV, V, I (3) They can cut DNA at variable site
(3) III, IV, V, I, II (4) II, III, V, IV, I (4) They can cut DNA at specific base sequences
Q.5 A eukaryotic gene has "sticky ends" produced by Q.15 Chimeric DNA is -
the restriction endonuclease EcoRI. The gene (1) DNA which contains uracil
is added to a mixture containing EcoRI and a bacterial (2) DNA sythesized from RNA
plasmid that carries two genes, which make it resistant (3) Recombinant DNA
to ampicillin and tetracyline. The plasmid that carries (4) DNA which contains single
two genes, which make it resistant to ampicillin and Q.16 A piece of nucleic acid using to find out a gene,
tetracycline. The plasmid has one recognition site for by forming hybrid with it, is called as-
EcoRl located in the tetracycline resistance gene. This (1) c-DNA (2) DNA probe
mixture is incubated for several hours and then added to (3) Sticky end (4) Blunt end
bacteria growting in nutrient broth. The bacteria are Q.17 Which of the following is the example of direct
allowed to grow overnight and are streaked on a plate gene transfer -
using a technique that produces isolated colonies that (1) Micronjection (2) Electroporation
are clones of the original. Samples of these colonies are (3) Particle gun (4) All the above
then grown in four different media: nutrient broth plus
33
Q.18 How many copies of DNA sample are produced in (a) Electrophoresis (b) Electroporation
PCR technique after 6-cycle - (c) Methylation (d) Restriction digestion
(1) 4 (2) 32 (1) a and c (2) c and d
(3) 16 (4) 64 (3) a and d (4) b and d
Q.19 The basic procedure involved in the synthesis of Q.23 Restriction enzymes are -
recombinant DNA molecule is depicted. The mistake in (1) Not always required in genetic engineering
the procedure is - (2) Essential tool in genetic engineering
(3) Nucleases that cleave DNA at specific sites
(4) (2) and (3) both
Q.24 Function of restriction endonuclease enzyme is-
(1) Useful in genetic engineering
(2) Protects the bacterial DNA against foreign DNA
(3) Helpful in transcription
(4) Helpful in protein synthesis
Q.25 Which of the following restriction endonuclease
enzyme produce blunt end in DNA -
(1) Enzyme polymerase is not included
(2) The mammalian DNA is shown double stranded
(3) Only one fragement is inserted
(4) Two different restricition enzymes are used (1) (2)
Q.20 Western blotting is used for the identification of -
(1) DNA (2) RNA
(3) Protein (4) All of the above (3) (4) All the above
Q.21 In r-DNA technique which of the following Q.26 A bacterium modifies its DNA by adding methyl
technique is not used in introducing DNA into host groups to the DNA, It does so to -
cell - (1) Clone its DNA
(1) Transduction (2) Conjugation (2) Be able to transcribe many genes simultaneously
(3) Transformation (4) Electroporation (3) Trun its gene on
Q.22 Which of the following techniques are used in (4) Protect its DNA from its own restriction enzyme
analyzing restriction fragment length polymorphism
(RFLP) -
Answer Key
Section–A
Q.1 3 Q.2 3 Q.3 2 Q.4 4 Q.5 1 Q.6 4 Q.7 4 Q.8 4 Q.9 1 Q.10 2 Q.11 1 Q.12 2 Q.13 2 Q.14 2
Q.15 4 Q.16 4 Q.17 3 Q.18 4 Q.19 4 Q.20 4 Q.21 3 Q.22 1 Q.23 1 Q.24 3 Q.25 2 Q.26 3 Q.27 2
Q.28 4 Q.29 3 Q.30 3 Q.31 2 Q.32 4 Q.33 4 Q.34 4 Q.35 4 Q.36 3 Q.37 3 Q.38 4 Q.39 2 Q.40 4
Q.41 3 Q.42 2 Q.43 4 Q.44 4 Q.45 4 Q.46 4 Q.47 2 Q.48 1 Q.49 2 Q.50 4
Section–B
Q.1 2 Q.2 4 Q.3 3 Q.4 2 Q.5 3 Q.6 3 Q.7 4 Q.8 3 Q.9 2 Q.10 1 Q.11 2 Q.12 2 Q.13 4 Q.14 4
Q.15 3 Q.16 2 Q.17 4 Q.18 3 Q.19 4 Q.20 3 Q.21 2 Q.22 3 Q.23 4 Q.24 2 Q.25 3 Q.26 4
34
Exercise–2 Previous Year's Questions
Q.1 Plasmid has been used as vector because (1) Bacterial cell can carry out the RNA spling
(1) It is circular DNA which have capacity to join to reactions
eukaryotic DNA (2) The mechanism of gene regulation is identical in
(2) It can move between prokarytic and eukaryotic cells humans and bacteria
(3) Both ends show replication (3) Human chromosome can replicate in bacterial cell
(4) It has antibiotic resistance gene (4) The genetic code is universal
Q.2 Which of the following cuts the DNA from specific Q.11 Electroporation procedure involves - [AIIMS-2005]
places - [AIPMT-2001] (1) Fast passage of food through sieve pores in phloem
(1) Restriction endonuclease (E-Co-RI) elements with the help of electric sitmulation
(2) Ligase (2) Opening of stomatal pores during night by artificial
(3) Exonuclease light
(4) Alkaline phosphate (3) Making transient pores in the cell membrane to
Q.3 Manipulation of DNA in genetic engineering introduce gene constructs
become possible due to the discovery of - (4) Purification of saline water with the help of a
(1) Restriction endonuclease (2) DNA ligase membrane system
(3) Transcriptase (4) Primase Q.12 The total number of nitrogenous bases in human
Q.4 Restriction enzymes - [AIIMS-2003] genome is estimated to be about -
(1) Are endonucleases which cleave DNA at specific sites [AIIMS - 2004]
(2) Make DNA complementary to existing DNA or RNA (1) 3.5 million (2) 35 thousand
(3) Cut or join DNA fragments (3) 35 million (4) 3.1 billion
(4) Are required in vectorless direct gene transfer Q.13 Two microbes found to be very useful in genetic
Q.5 DNA fingerprinting refers to - [AIPMT-2004] engineering are - [AIPMT - 2006]
(1) Techniques used for identification of fingerprints of (1) Escherichia coli and Agrobacterium tumefaciens
individuals (2) Vibro cholerae and a tailed bacteriophage
(2) Molecular analysis of profiles of DNA samples (3) Diplococcus sp. and Pseudomonas sp.
(3) Analysis of DNA samples using imprinting devices (4) Crown gall bacterium and Caenorhabdits elegans
(4) Techniques used for molecular analysis of different Q.14 Restriction endonuclease - [AIPMT-2006]
specimens of DNA (1) Cuts the DNA molecule randomly
Q.6 Restriction endonucleases - [AIPMT-2004] (2) Cuts the DNA molecule at specfic sites
(1) Are synthesized by bacteria as part of their defence (3) Restricts the synthesis of DNA inside the nucleus
compound. (4) synthesizes DNA
(2) Are present in mammalian cells for degradation of Q.15 The restriction enzyme ECO RI has the property
DNA when the cell dies of - [AMU -2007]
(3) Are used in genetic engineering for ligating two (1) Endonuclease activity
DNA molecules (2) Exonuclease activity
(4) Are used for invitro DNA sythesis (3) Ligation activity
Q.7 What is the first step in the Southern Blot techique- (4) Correcting the topology of replicating DNA
[AIIMS-2004] Q.16 DNA ligase is an enzyme that catalyses the-
(1) Denaturation of DNA on the gel for hybridization [AMU -2007]
with specific probe (1) Splitting of DNA threads into small bits
(2) Production of a group of genetically identical cells (2) Joining of the fragments of DNA
(3) Digestion of DNA by restriction enzyme (3) Denaturation of DNA
(4) Isolation of DNA from a nucleated cell such as the (4) Synthesis of DNA
one from the scene of crime Q.17 More advancement in genetic engineering is due
Q.8 Which of the following is not produced by E.Coil to - [Jharkhand - 2006]
in the lactose - (1) Restriction endonuclease (2) Reerse transcriptase
(1) galactosidase (3) Protease (4) Zymase
(2) Thiogalactoside transacetylase Q.18 The function of polymerase chain reaction (PCR) is -
(3) Lactose dehydrogenase (1) translation (2) transcription
(4) Lactose permease (3) DNA amplification (4) None of these
Q.9 The technique of transferring DNA fragement Q.19 Which of the following is used as a best genetic
separated on agarose gel to a synthetic membrane such vector - [MP PMT - 2007]
as nitrocellulose is known as- [OLYMPIAD-2004] (1) Bacillus thuriengenesis
(1) Northern blotting (2) Southern blotting (2) Agrobacterium tumifaciens
(3) Western blotting (4) Dot blotting (3) Pseudomonas putida
Q.10 Production of a human protein in bacteria genetic (4) All of these
engineering is possible because – [AIPMT-2005] Q.20 The transfer of protein from electrophoretic gel to
nitrocellulose membrane is known as-
35
[MP PMT - 2003] Q.31 Biolistics (gene-gun) is suitable for : [AIPMT
(1) transferase (2) northen blotting Mains 2012]
(3) westen blotting (4) southern blotting (1) Disarming pathogen vectors
Q.21 DNA - finger printing was first discovered by- (2) Transformation of plant cells
[MP PMT - 2007] (3) Constructing recombinant DNA by joining with
(1) Alec jeffery (2) Cark mullis vectors
(3) C. Milstein (4) Dr. Paul Berg (4) DNA finger printing
Q.22 Which of the following enzyme is used to join Q.32 In genetic engineering, the antibiotics are used :
DNA fragments - [MP PMT - 2003] [AIPMT Mains 2012]
(1) Terminase (2) Endonuclease (1) as selectable markers
(3) Ligase (4) DNA polymerase (2) to select healthy vectors
Q.23 Restriction endonucleases are enzymes which – (3) as sequences from where replication starts
(1) make cuts at specific positions within the DNA (4) to keep the cultures free of infection
molecule Q.33 What is it that forms the basis of DNA
(2) recognize a specific nucleotide sequence for binding Fingerprinting? [AIPMT Mains 2012]
of DNA ligase (1) The relative proportions of purines and pyrimidines
(3) restrict the action of the enzyme DNA polymerase in DNA
(4) remove nucleotides from the ends of the DNA (2) The relative difference in the DNA occurrence in
molecule blood, skin and saliva
Q.24 Satellite DNA is useful tool in – [CPMT - 2010] (3) The relative amount of DNA in the ridges and
(1) Organ transplantation (2) Sex determination grooves of the fingerprints.
(3) Forensic science (4) Genetic engineering (4) Satellite DNA occurring as highly repeated short
Q.25 PCR and Restriction Fragment Length DNA segments
Polymorphism are the methods for : [AIPMT Pre 2012] Q.34 The figure below shows three steps (A, B, C) of
(1) Genetic transformation (2) DNA sequencing Polymerase Chain Reaction (PCR). Select the option
(3) Genetic Fingerprinting (4) Study of enzymes giving correct identification together with what it
Q.26 Which one is a true statement regarding DNA represents? [AIPMT Mains 2012]
polymerase used in PCR? [AIPMT Pre 2012] Options :
(1) It serves as a selectable marker (1) B-Denaturation at a temperature of about 98ºC
(2) It is isolated from a virus separating the two DNA strands.
(3) It remains active at high temperature (2) A - Denaturation at a temperature of about 50ºC
(4) It is used to ligate introduced DNA in recipient (3) C-Extension in the presence of heat stable DNA
cells. polymerase
Q.27 For transformation, micro-particles coated with (4) A - Annealing with two sets of primers
DNA to be bombarded with gene gun are made up of : Q.35 Which one of the following repreents a
[AIPMT Pre 2012] palindromic sequence in DNA? [AIPMT Mains 2012]
(1) Platinum or Zinc (2) Silicon or Platinum (1) 5’ - GAATTC - 3’; 3’ - CTTAAG - 5’
(3) Gold or Tungesten (4) Silver or Platinum (2) 5’- CCATCC-3’; 3’ - GAATCC - 5’
Q.28 A single strand of nucleic acid tagged with a (3) 5’ - CATTAG - 3’; 3’ - GATAAC - 5’
radioactive molecule is called : [AIPMT Pre 2012] (4) 5’ - GATACC - 3’; 3’ - CCTAAG - 5’
(1) Selectable marker (2) Plasmid Q.36 DNA fragments generated by the restriction
(3) Probe (4) Vector endonucleases in a chemical reaction can be separated
Q.29 The figure below is the diagrammatic by : [AIPMT 2013]
representation of the E.Coli vector pBR 322. Which one (1) Polymerase chain reaction (2) Electrorhoresis
of the given options correctly identifies its certain (3) Restriction mapping (4) Centrifugation
component (s)? [AIPMT Pre 2012] Q.37 The colonies of recombinant bacteria appear white
(1) rop-reduced osmotic pressure in contrast to blue colonies of non-recombinant bacteria
(2) Hind III, EcoRI - selectable markers because of : [AIPMT 2013]
(3) ampR, tetR - antibiotic resistance genes (1) Insertional inactivation of alpha-galactosidase in
(4) ori - original restriction enzyme non-recombinant bacteria
Q.30 Which one of the following is a case of wrong (2) Insertional inactivation of alpha-galactosidase in
matching ? [AIPMT Pre 2012] recombinant bacteria
(1) Vector DNA - Site for t-RNA synthesis. (3) Inactivation of glycosidase enzyme in recombinant
(2) Micropropagation - In vitro production of plants in bacteria
large numbers. (4) Non-recombinant bacteria containing beta-
(3) Callus - Unorganised mass of cells produced in galactosidase
tissue culture. Q.38 In vitro clonal propagation in plants is
(4) Somatic hybridization - Fusion of two diverse cells. characterized by : [AIPMT 2014]
(1) Electrophoresis and HPLC
36
(2) Microscopy Q.40 Which vector can clone only a small fragment of
(3) PCR and RAPD DNA? [AIPMT 2014]
(4) Northern blotting (1) Plasmid
Q.39 An analysis of chromosomal DNA using the (2) Cosmid
Southern hybridization technique does not use: (3) Bacterial artificial chromosome
[AIPMT 2014] (4) Yeast artificial chromosome
(1) Autoradiography (2) PCR
(3) Electrophoresis (4) Blotting
Answer Key
Q.1 1 Q.2 1 Q.3 1 Q.4 1 Q.5 2 Q.6 1 Q.7 1 Q.8 3 Q.9 2 Q.10 4 Q.11 3 Q.12 4 Q.13 1 Q.14 2
Q.15 1 Q.16 2 Q.17 1 Q.18 3 Q.19 2 Q.20 3 Q.21 1 Q.22 3 Q.23 1 Q.24 3 Q.25 3 Q.26 3 Q.27 3
Q.28 3 Q.29 3 Q.30 1 Q.31 3 Q.32 1 Q.33 4 Q.34 3 Q.35 1 Q.36 2 Q.37 4 Q.38 3 Q.39 2 Q.40 1
Exercise–3 AIIMS Special Questions

Assertion -Reason Type Questions
In the following questions, a statement of assertion (A) is followed by a statement of reason (R).
(1) If both Assertion & Reason are true and the reason is the correct explanation of the assertion,
then mark (1).
(2) If both Assertion & Reason are true but the reason is not the correct explanation of the assertion,
then mark (2).
(3) If Assertion is true statement but Reason is false, then mark (3).
(4) If both Assertion and Reason are false statements, then mark (4).
Q.1 A : DNA ligase play important role in recombinant DNA technology.
R : The linking of antibiotic resistance gene with plasmid vector became possible by enzyme DNA
ligase.
Q.2 A : Restriction enzymes belong to a larger class of enzymes called nucleases.
R : Each restriction enzyme recognises a specific palindromic nucleotide sequence in the DNA.
Q.3 A : During gel electrophoresis, the DNA fragments move towards the anode.
R : DNA fragments are negatively charged molecules.
Q.4 A : Selection of recombinants due to inactivation of antibiotics is cumbersome procedure.
R : It requires simultaneous plating on two plates having different antibiotics.
Q.5 A : Taq Polymerase is involved in PCR technique.
R : This enzyme remain active during the high temperature including denaturation of double
stranded DNA.
Q.6 A : Small DNA fragments will arrange towards positive end after the gel electrophoresis in
DNA test.
R : DNA is negatively charged.
Q.7 A : PCR-technique is used in amplification of specific gene.
R : In PCR-technique Taq-polymerase enzyme is used and this enzyme is thermo-sensitive.
Answer Key
Q.1 1 Q.2 2 Q.3 1 Q.4 1 Q.5 1 Q.6 1 Q.7 3
37

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BIOLOGY-XII BIOTECHNOLOGY 1

Biotechnology is use of microorganisms, plant, animal cells, their components or enzymes

ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

Haomophilus 5’-G-G- C-C-3’

Streptomyces 5’-A-G-T- A-C-T-3’

ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

ADDRESS - Plot No. 1191, Sector-5, Vasundhara, Ghaziabad. PHONE – 0120-4545-326.

Parameter PCR Gene cloning

Brief History and Development of Genetic Engineering

Flavr Savr’ Tomato - A GM plant – (Maintain Post harvesting loss)

Exercise–3 AIIMS Special Questions

Q.1 1 Q.2 2 Q.3 1 Q.4 1 Q.5 1 Q.6 1 Q.7 3

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