BIOTECHNOLOGY:

PRINCIPLES, PROCESSES
AND
APPLICATIONS
BY
B.H.SOORYA RAO
CLASS XII

DEFINITION

Biotechnology deals with techniques of using live organisms

or their components or enzymes from organisms to produce
products and processes useful to humans.

The European Federation of Biotechnology (EFB) furnishes

its implication as:
The integration of natural science and organisms, cells,
parts thereof, and molecular analogues for products and
services.

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TECHNIQUES OF GENETIC ENGINEERING

The technique of genetic engineering includes :
(i) Creation of recombinant DNA
(ii) Use of gene cloning, and
(iii) Gene transfer. With this technique, we can
isolate and introduce only the desired genes
without introducing undesirable genes into
target organisms.

WHAT IS CLONING?
Cloning or making multiple copies of
any template DNA needs that an alien
DNA is linked with the origin of
replication (a desired DNA sequence
responsible for initiating replication) so
that alien (foreign/desired) pieces of
DNA can replicate and multiply itself in
the host organism.

CREATION OF RECOMBINANT DNA

The first recombinant DNA was constructed by Stanley Cohen and
Herbert Boyer in 1972 by;
(i) Isolating the antibiotic resistance gene by cutting out a piece of
DNAfrom a plasmid which was responsible for providing antibiotic
resistance. The cutting of DNA at specific location was done by
molecular scissors restriction enzymes.
(ii) The cut pieces of DNA were then linked with the plasmid DNA.
(iii) The plasmid DNA acts as vector to transfer the pieces of DNA attached
to it, to the recipient host bacterium.
(iv) Linking of antibiotic resistant gene (alien DNA) with plasmid DNA
(vector) was possible with enzyme DNA ligase, which joins the cut
ends of DNA molecules.
(v) Hence, a new circular, autonomously replicating DNA created in vitro is
formed called as recombinant DNA.
(vi) It replicates using the new hosts DNA polymerase enzyme and makes
multiple copies. The ability to multiply copies of antibiotic resistant
gene in E. coli is called cloning.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

(i) Restriction enzymes

(ii) Polymerase enzymes
(iii)Ligases
(iv)Vectors
(v)Host organisms

RESTRICTION ENZYMES

Restriction enzymes belong to a class of enzymes

called nucleases. They are of two types:
(a) Exonucleases : Remove nucleotides from the
ends/terminals of DNA.
(b) Endonucleases : Cut the DNA at specific
position of N-bases anywhere in the length except
ends.

RESTRICTION ENDONUCLEASESAN INTRODUCTION

Linn and Arber (1963) isolated two enzymes from E. coli

responsible for restricting the growth of bacteriophage, one
of them added methyl group (CH3 -) to DNA while the other
cut the DNA into segments and is called restriction
endonucleases.

Smith, Willox and Kelley (1968) isolated and

characterized the first restriction endonuclease from
Haemophilus influenzae bacterium called it Hind-II. HindII always cut DNA at a particular site/point by recognizing
a specific sequence of six base pairs called recognition
sequence for Hind-II. Today more than 900 restriction
enzymes have been isolated from over 200 strains of
bacteria, each of which recognizes different recognition
sequences.

RESTRICTION ENDONUCLEASES (CONTD)

The recognition sequence is palindromic where the sequence of base pairs

reads the same on both the DNA strands when the orientation of reading is
kept the same, i.e., 53 or 35 direction e.g. 5- GAATTC- 3 3- CTTAAG
-5.
Each RE function by inspecting the length of a DNA and then binds to the
DNA at recognition sequences.
RE cut the two strands of DNA double helix at specific points in their sugarphosphate backbone, a little away from the centre of palindromic site, but
between the same two bases on both the strands. As a result single stranded
portions are left at the end of DNA in each strand called sticky ends.
Unwanted self ligation of vector DNA molecules by removing phosphate
group from the 5 end of a DNA molecule, leaving a free 5 hydroxyl group,
using alkaline phosphate from bacteria (BAP) or calf intestine (CAP).
These single stranded DNAs in each strand form hydrogen bonds with their
complementary counterpart (single strands of alien DNA). This stickiness of
the ends facilitates the action of enzyme DNA ligase. Hence RE are used in
genetic engineering to form Recombinant molecules of DNA

RESTRICTION ENDONUCLEASES (contd)

When cut by the same RE, the resultant DNA fragments have the same
kind of sticky ends and these can be joined together using DNA ligase.

NOMENCLATURE OF RESTRICTION
ENZYMES

The first letter of the name comes from genus and the next two
letters from the name of the species of the species of the bacterium
(prokaryotic cell) from which they are isolated.

The next letter comes from the strain of the bacterium.

The Roman number following these four letters indicate the order in
which enzymes were isolated from that strain of the bacterium, e.g.
(a) Eco R-I is isolated from Escherichia coli RY 13
(b) Hind- II is from Haemophilus influenzae
(c) Bam H-I is from Bacillus amyloliquefaciens
(d) Sal-II is from Streptomyces albus.

CLONING VECTOR

Cloning Vector includes plasmid and bacteriophages, which have

the ability to replicate within bacterial cells and independent of
the control of chromosomal DNA. A vector should have very high
copy number of their genome within their bacterial cell so that a
linked alien piece of DNA, can multiply its number equal to the
copy number by vector employed.

Vectors used at present are engineered in such a way that they

help easy linking of foreign/alien DNA and selection of
recombinant from non-recombinant.

CLONING VECTOR

FEATURES OF VECTOR

Following are the features that are required to facilitate cloning in

a vector.\
1. Origin of replication
2. Selectable marker
3. Cloning site (Recognisation vector)
4. Small size of vector

Origin of replication (ori): This is the sequence from where

replication starts and a piece of DNA linked to this sequence can
be made to replicate within host cells. This sequence also controls
the copy number of vector DNA or linked alien DNA.

Selectable marker : It helps in identifying ang eliminating nontransformants and help in selecting those host cells which contain
the vector, i.e., transformants.

Cloning sites : The vector should have a few or atleast one

VECTORS FOR CLONING GENES IN

PLANTS AND ANIMALS
In plants, the Ti-plasmid (Tumour inducing plasmid)
of bacterium Agrobacterium tumefaciens has been
modified (does not cause tumour ) is used as a
cloning vector .
Similarly, retroviruses also make the normal animal
cells into cancerous cells. The retroviruses have also
been disarmed and are now used to deliver desirable
genes into animal cells.
Once a gene or a DNA fragment has been ligated into
a vector, it is transferred into a bacterial, plant or
animal host.

PROCESSES OF RECOMBINANT DNA

TECHNOLOGY
IT INVOLVES THE FOLLOWING STEPS :
(i) Isolation of DNA
(ii) Fragmentation of DNA by REs
(iii) Isolation of desired DNA fragment
(iv) Amplification of the gene of interest
(v) Ligation of DNA fragment into vector
(vi) Transferring the recombinant DNA into host
(vii) Culturing the host cells in a culture medium at large scale
(viii) Extraction of desired products.

PROCESSES OF
RECOMBINANT DNA
TECHNOLOGY
IN DETAIL

1.

ISOLATION OF DNA
DNA is enclosed by membranes along with other macromolecules such as
RNA, proteins polysaccharides and lipids.
(a) The membrane is dissolved by treating the bacterial cells/plant or animal
tissue with enzymes such as lysozyme (bacteria), cellulase (plant cell),
chitinase (fungus).
(b) Long DNA molecules are interwined with histone proteins. RNA can be
removed by treating with ribonuclease whereas the proteins can be removed
by proteases.
(c) Other molecules can be removed by treating by appropriate treatments
and now purified DNA precipitates out after the addition of chilled ethanol.
(d) It is seen as collection of fine thread in the suspension.

DNA that separates out can be removed by spooling

2.

CUTTING DNA AT SPECIFIC LOCATION

(a) Fragmentation of DNA is carried out by incubating purified DNA
molecules with restriction enzyme at optimal conditions of temperature and pH
for that specific enzyme.
(b) Agarose gel electrophoresis technique is employed to check the
progression of restriction enzyme digestion.
(c) The similar process is repeated with vector DNA.

3. SEPARATION AND
ISOLATION OF DNA
FRAGMENTS

(a) DNA fragments being negatively charged,

can be separated by forcing them to move
towards anode under an electric field through
a medium/matrix (agarose). The smaller the
fragment size, the farther it moves.
(b) DNA fragments can be visualized by
staining by staining DNA with ethidium
bromide followed by exposure to UV
radiations. Bright orange colour bands in
DNA became prominent in the gel. The
separated bands of DNA are cut out from gel
and extracted from the gel piece. This step is
known as elution.
(c) Purified DNA fragments are used for
reconstructing recombinant DNA by joining
them with cloning vectors.

4. AMPLIFICATION OF GENES OF INTEREST USING PCR

a)

PCR stands for polymerase chain reaction. In this reaction, multiple

copies of gene /DNA of interest are synthesised in vitro using two sets of
primers.
b) The enzyme DNA polymerase.
c) The enzyme extends the primer using the nucleotides provided in the
reaction and the genomic DNA as template.
d) Continued DNA replication of segment of DNA can be amplified to
approximately 1 billion times i.e., 1 billion copies are made.
e) Repeated replication is possible by the use of thermostable DNA
polymerase (isolated from bacterium Thermus aquaticus ) which remains
active during high temperature induced denaturation o double stranded
DNA.
f) Amplified fragment can be used to ligate with vector for further cloning.

5.

JOINING OF DNAs
(a) After cutting DNA with specific RE, the cut out gene of interest from the
source DNA and the cut vector with space are mixed and ligase is added.
(b) This results in the preparation of recombinant DNA.

6.

INSERTION OF RECOMBINANT DNA INTO HOST

CELL/ORGANISM
There are several methods of introducing the ligated DNA into recipient
cells.
Recipient cells after making them competent to receive, take up DNA
present in its surrounding. So, if a recombinant DNA bearing gene
for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli
cells, the host cells become transformed into ampicillin-resistant cells. If
we spread the transformed cells on agar plates containing ampicillin, only
transformants will grow, untransformed recipient cells will die. Since, due
to ampicillin resistance gene, one is able to select a transformed cell in the
presence of ampicillin. The ampicillin resistance gene in this case is called
a selectable marker.

7. OBTAINING THE FOREIGN GENE PRODUCT

Ultimate aim of all recombinant technologies is to produce a
desirable proteins by expression of recombinant DNA. The foreign
gene gets expressed under suitable conditions, by culturing the host
cell on a suitable medium.

RECOMBINANT PROTEIN
Recombinant protein is produced if any protein encoding
gene is expressed in heterologous host.
(i) The transformed cells containing cloned gene of interest
are grown in cultures

BIOREACTORS
Bioreactors are used for processing large volume of
culture for obtaining the product of interest in large
quantities. In bioreactors the raw materials are
converted into specific products, e.g., enzymes.
A bioreactor provides the optimal conditions for
achieving the desired product by providing optimal
growth condition such as:
(i) temperature
(ii) pH
(iii) substrate
(iv) salt
(v) vitamins
(vi) oxygen

BIOREACTORS

APPLICATIONS OF
BIOTECHNOLOGY

REASEARCH AREAS IN
BIOTECHNOLOGY
Three critical research areas of biotechnology are :

(i) Providing the best catalyst in the form of improved organism

usually a microbe or pure enzyme.
(ii) Creating optimal conditions through engineering for a catalyst
to act and
(iii) Downstream processing technologies to purify the
protein/organic compound.

BIOTECHNOLOGICAL APPLICATIONS IN
AGRICULTURE
Three options for increasing food production :
1. Agrochemical based agriculture
2. Organic agriculture
3. Genetically engineered crop based agriculture.

Green Revolution has succeeded in tripling the yield of crops due

to :
(i) Improved crop varieties
(ii) Use of better management practices
(iii) Use of agrochemicals, i.e. fertilisers, insecticides and pesticides
etc.

GENETICALLY MODIFIED ORGANISMS

(GMO)
GM plants are useful in many ways. Some of their unique features are:
1. More tolerant to abiotic stresses such as cold, drought, etc.
2. Have reduced dependence on chemical pesticides (pest-resistant
crops).
3. Reduction in post harvest losses.
4. Increased efficiency of mineral usage by plants.
5. Enhanced nutritional value of food, e.g. Golden rice and sweet potato.
6. Used to create tailor made plants to supply alternative sources to
industries in the form of starches, fuels and pharmaceuticals.
7. Production of pest-resistant plants, e.g. Bt-cotton, Bt-corn, rice and
soyabean etc.

PEST - RESISTANT PLANTS

Several nematodes parasitise a wide variety of plants and
animals including human beings.
1.

A nematode Meloidegyne incognitia infects the roots of tobacco plants and

causes a great reduction in yield.

2.

A novel strategy was adopted to prevent this infestation which was based
on the process of RNA interference (RNAi). RNAi takes place in all
eukaryotic organisms as a method of cellular defense. This method
involves silencing of a specific mRNA due to a complementary dsRNA
molecule that binds to and prevents translation of the mRNA (silencing).

3.

The source of this complementary RNA could be from an infection by

viruses having RNA genomes or mobile genetic elements (transposons)
that replicate via an RNA intermediate.

4.

Using Agrobacterium vectors, nematode-specific genes were introduced

into the host plant .

5.

The introduction of DNA was such that it produced both sense and antisense RNA in the host cells.

6.

These two RNAs being complementary to each other formed a double

stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA
of the nematode.

7.

The consequence was that the parasite could not survive in a transgenic
host expressing specific interfering RNA.

8.

The transgenic plant therefore got itself protected from the parasite

GENE THERAPY

Gene therapy is a collection of methods that allow correction of a gene defect

that has been diagnosed in a child/embryo required a mechanism to switch off a
defective gene and to substitute a healthy gene copy.
The first clinical gene therapy was given in 1990 to a 4-year old girl with
adenosine deaminase (ADA) deficiency. This enzyme is crucial for
the immune system to function.
The disorder is caused due to the deletion of the gene for 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 the problem with both of these approaches that they are not completely
curative.
As a first step towards gene therapy, lymphocytes from the blood of the patient
are grown in a culture outside the body.
A functional ADA cDNA (using a retroviral vector) is then introduced into these
lymphocytes, which are subsequently returned to the patient. However, as
these cells are not immortal, the patient requires
periodic infusion of such genetically engineered lymphocytes. However, if
the gene isolate from marrow cells producing ADA is introduced into cells
at early embryonic stages, it could be a permanent cure.

MOLECULAR DIAGNOSIS
Recombinant DNA Technology, polymerase chain reaction
(PCR), and Enzyme-linked Immunosorbent Assay (ELISA) are
some of techniques diagnosis because :
1. Very low concentration of bacteria or virus (before appearance of visible
symptoms of disease) can be detected by amplification of their nucleic acids.
2. PCR is now used to detect HIV in suspected AIDS patients.
3. A probe is a piece of single stranded DNA that is tagged with a radioactive
molecule and it is used to find it complementary by hybridization.
4. Presence of normal or mutated gene can be detected by this method .

TRANSGENIC ANIMALS
Transgenic animals are those that have had their DNA manipulated to
posses and express an extra or foreign gene, example transgenic rats, rabbits,
pigs, sheep, cow, etc.

Reasons for genetical modification of animals

1. Normal physiology and development
2. Study of disease
3. Biological products
4. Vaccine safety
5. Chemical safety testing

ETHICAL ISSUES

Modification of living organisms by human without

regulation and some ethical standards cannot be
allowed.

The modifications and uses of living organisms for

public service have resulted in problems with the
granting of patents for them.

Hence, Govt. of India has set up organization such

as GEAC (Genetic Engineering Approval
Committee) which will make decisions regarding
the validity of GM research and the safety of
introducing GM organisms for public service.

BIOPIRACY
Biopiracy is the term used to refer to the use of bioresources
by MNCs and other organizations without proper authorization
from the country and people concerned without compensatory
payment.
(i) Financially rich industrialized nations but poor in biodiversity
exploit the bioresources and traditional knowledge of developing and
underdeveloped poor countries rich in biodiversity.
(ii) Some nations are developing laws to prevent such unauthorized
exploitation of their bioresources and traditional knowledge.

SOME WONDERFUL FACTS

mouse with human ear