Biotechnology :

Principles and
Processes

Prepared by:
Mrs. Priyanka Tyagi
PGT (Biology)
K.V. Dogra Lines Meerut
Cantt
What is biotechnology?
 Biotechnology refers to the
technology using biology, which
has applications in agriculture,
food processing industry,
medicine diagnostics,
bioremediation, waste treatment,
and energy production.
 Biotechnology
 Biotechnology deals with the techniques of
using live organisms or enzymes from
organisms to make products and processes
that benefit human beings.
 The definition given by European Federation of
Biotechnology (EFB) is as follows:
“The integration of natural science and
organisms, cells, parts thereof, and molecular
analogues for products and services.”
 Principles of Biotechnology
 Genetic engineering : Introduction of
foreign genetic material (DNA/RNA)
into the host’s genome and altering its
phenotype.
 Aseptic techniques : Maintenance of
sterile atmosphere to enable growth
of only the desired cell in large
quantities for the manufacture of
products like antibiotics, vaccines
enzymes etc.
 Techniques Of Genetic
Engineering
 Creation of recombination DNA
 Gene transfer into host
organism
 Gene cloning
Creation Of Recombination
DNA
 Stanley Cohen and Herbert Boyer
(1972) constructed the first
recombination DNA.
 They isolated the antibiotic resistance
gene from the Plasmid of the bacterium
Salmonella typhimurium.
 This piece of DNA carrying antibiotic
resistance gene was cut at specific
location by restriction endonuclease,
popularly known as Molecular Scissors.
Gene transfer

 The cut piece of DNA was introduced

in the plasmid of Escherichia coli
which acted as the vector.
 The piece of DNA was ligated to the
vector plasmid by DNA ligase.
 This joining of the two DNA pieces
resulted in the creation of
recombinant DNA.
Gene cloning
 The new recombinant DNA was
transferred into E. coli.
 The r DNA replicated autonomously
by using the host DNA polymerase
enzyme and made multiple copies.
 The ability to multiply copies of any
template of DNA is called gene
cloning.
Tools of Recombinant
DNA Technology
Restrictionenzymes
Cloning Vector
DNA polymerase enzyme
DNA Ligase enzyme
Host organism
Restriction enzymes

As
biological
scissors
!!!!
Restriction enzymes
 Restriction enzmes belong to a class
of enzymes called nucleases.
 These are of two types – exonucleases
and endonucleases.
 Exonucleases cut the DNA at the
ends.
 Endonucleases make cuts at specific
points in the DNA.
Restriction enzymes

 The first restriction endonuclease

isolated – Hind II.
 It was isolated from the bacterium
Haemophilus influenzae.
 Today we know more than 900
restriction enzymes isolated from 230
strains of bacteria.
Recognition sequence
 Restriction enzymes always cut DNA
molecules at a particular point by
recognizing a specific sequence of base
pairs.
 This specific base sequence is known as
recognition sequence.
 Example: EcoRI recognises only the
following sequence.
5’-------GAATTC-------3’
3’-------CTTAAG-------5’
 Restriction Enzymes…….How do you
denote them????
• Names use 3-letter italicized code:
 1st letter - genus
 2nd & 3rd - species
• Following letter denotes strain

EcoRI
was the first restriction enzyme found in the R strain of E. coli
Palindromic Sequence
 Palindromes are those group of letters
which read the same, both forward and
backward. Ex. MALYALAM
 A Palindromic sequence is a sequence
which reads the same on the two
strands of DNA when orientation is
kept the same.
5’-------GAATTC-------3’
3’-------CTTAAG-------5’
Cutting & Pasting…….Restriction enzymes as
molecular scissors
 Restriction enzymes
 Three types: Types I, II and III

 Type II used for molecular biology work

 Endonucleases

 Recognize specific sequence of base-pairs,

usually 4, 6 or 8 bases that are palindromic

 Can leave ‘sticky ends’ or ‘blunt ends’

 About sticky and blunt ends………
5’ ATGCGAATTCCGGAA 3’
3’ TACGCTTAAGGCCTT 5’

EcoR1 Sticky ends

5’-ATGCG-3’ 5’-AATTCCGGAA-3’
3’-TACGCTTAA-5’ 3’-GGCCTT-5’

5’ ATGCGATATCCGGAA 3’
3’ TACGCTATAGGCCTT 5’

EcoRV
Blunt ends
5’-ATGCGAT-3’ 5’-ATCCGGAA-3’
3’-TACGCTA-5’ 3’-TAGGCCTT-5’
 What do restriction enzymes
help us achieve ???
• They help in generating DNA fragments with precise ends

• These precisely generated ends can then be ‘pasted’ to similar

ends in a vector molecule that is self replicating.

• The process of ‘pasting’ is called ligation and requires a DNA
ligase
• The product after ligation is called a ‘recombinant DNA
molecule’ or a ‘clone’
• Each clone can be replicated to provide ample material for study
Cloning vector is a DNA molecule that
carries foreign DNA into a host cell,
replicates inside a bacterial (or yeast) cell
and produces many copies of itself and the
foreign DNA.
Features of cloning vectors
Types of Cloning Vectors
General steps of cloning any vector
Features of Cloning Vectors
1. Origin of replication: a sequence from
where replication starts.
2. Selectable marker: a method of selecting
for bacteria containing a vector with
foreign DNA; permits the growth of
transformants and eliminate the non-
transformants. usually accomplished by
genes encoding resistant to antibiotics
such as ampicillin,
chloramphenicol,tetracycline or
kanamycin.
3. Cloning site: to insert foreign DNA; the
most versatile vectors contain a site that
It shows a typical plasmid vector. It contains a polylinker which can recognize
several different restriction enzymes, an ampicillin-resistance gene (ampr) for
selective amplification, and a replication origin (ORI) for proliferation in the host
cell.
 Plasmids or Bacteriophages are
used as vectors.
 If we are able the link an alien
DNA with Bacteriophage or
Plasmid DNA, we can multiply its
number equal to the copy number
of the Plasmid or Bacteriophage.
Vectors
 DNA molecules capable of accepting a
‘foreign/new’ DNA fragment
 Most common are plasmids but could be of
other types also.
 They are self replicating because they have
an origin of replication (ori)
 Plasmids are circular, double-stranded, extra-
chromosomal pieces of DNA
 Plasmids carry antibiotic resistance genes and
a region to clone the ‘new’ DNA called the
multiple cloning site
PLASMID
 A plasmid is an independent, circular,
self-replicating DNA molecule that
carries only a few genes.
 Vectors for cloning genes in
plants
•Agrobacterium tumefaciens can cause the plant disease
crown gall by transferring specific genes to the dicot
plant.
•A. tumefaciens contains a large plasmid called Ti
plasmid which can deliver T- DNA to transform normal
cell into a tumor and direct these tumor cells to produce
the desired chemicals.
•Plant genetic engineers have used this natural
transformation system as a vehicle for the introduction
of foreign DNA into plants.
 So what is Agrobacterium?????

A natural genetic engineer!!

Agrobacterium tumefaciens
is a soil bacteria that causes
common plant tumours, commonly
known as Crown Gall disease which
affects a wide variety of plants.

The genome of Agrobacterium tumefaciens C58 has been

sequenced completely and consists of a circular chromosome,
a linear chromosome and two plasmids
A. tumefaciens gall is not a tiny thing!!!!!
 T-DNA
region

The Ti
Tumor-
Plasmid
producing
genes

Opine
catabolism

Virulence region

ORI
During infection, the Ti plasmid is integrated
into the plant chromosomal DNA
 Produce callus  transform callus 
stimulate shooting by cytokinin addition

+ cytokinin

This procedure is easy

for dicotyledon plants
(legumes etc)

Biology of Plants, Raven et. al., Freeman Worth Publishing, 1999

 Monocotyledons are not easy to handle –
callus is very difficult to be initiated, and
A.tumefaciens is not pathogenic for them
1. Pericarp sholud be pulled back and
the immature embryos (0.5 - 1.0 mm) are
removed.

2. The immature embryos

are placed on
a callus induction medium

Transformation
is performed
by gene gun method high osmotic media
prepare calli
for transfomation
After shooting calli are placed on a selective
media containing a herbicide for three weeks.

Then calli are transferred to a media

to induce the production of shoots.
After they form small shoots,
they are transferred to
DARKER containers on a root induction media.
Vectors for cloning genes in
Animals
 Retroviruses in animals have the ability to
transform the normal cells into cancerous
cells.
 Retroviruses are disarmed and now used to
deliver desirable genes into animal cells.
 Competent Host
(for transformation with r-DNA)
 Chemical treatment: Bacteria cell is treated by
calcium to make them competent.
 Incubate the cells with r-DNA on ice,
 heat shock by placing them at 42oC,
 putting them back on ice.
This enables the bacteria to take up the r-DNA.
Other methods
 Micro injection: r-DNA is directly injected into
the nucleus of an animal cell.
 Gene gun or Biolistics: plant cells are
bombarded with high velocity micro-particles
of gold or tungstun coated with r-DNA.
 Disarmed Pathogen: agrobacterium
tumifaciens in plants and Retroviruses in
animals.
Micro-injection: The host cell is immobilized by
applying a mild suction with a blunt pipette. The foreign
gene is then injected with a micro-injection needle.
 “Gene Gun” Technique
DNA coated Cell’s DNA
golden particles

Plant cell
Gene gun

A plant cell with

the new gene

Transgenic plant Cell division

DNA with desired gene and antibiotic resistance is
coated onto the surface of gold particles.

Calli are placed

in vacuum chamber,
Helium pressure
shot DNA into cells

Gene gun
Coating gold
particles with
DNA

vacuum chamber

Calli remain
on the high osmotic media
for 20 hours
following shooting.
Closer look on: “gene gun”
 Processes of Recombinant
DNA Technology
 Isoaltion of the Genetic Material(DNA)
 Cutting of DNA at specific location
 Amplication of Gene of Interest using
PCR
 Insertion of r-DNA into the Host
cell/organism
 Obtaining the Foreign Gene Product
Isoaltion of the Genetic Material(DNA)
 Construction of a
Recombinant DNA
 Plasmid (autonomously replicating, circular,
extra-chromosomal DNA) is isolated.
 Plasmid DNA acts as a vector since it is used
to transfer the piece of DNA attached to it
to the host.
 Plasmid DNA also contains genes responsible
for providing antibiotic resistance to the
bacteria.
 Plasmid DNA was cut with a specific
restriction enzyme (‘molecular scissors’ −
that cut a DNA at specific locations).
 The DNA of interest (to be inserted) was
also cut with the same restriction enzyme.
 The DNA of interest is hybridised with the
plasmid with the help of DNA ligase to
form a Recombinant DNA.
 Recombinant DNA is then transferred to a
host such as E.coli, where it replicates by
using the host’s replicating machinery.
 When E.coli is cultured in a medium
containing antibiotic, only cells containing
recombinant DNA will be able to survive
due to antibiotic resistance genes and one
will be able to isolate the recombinants.
Selecting cloned DNA molecules and
making more of them

Transformation and selection Plasmid multiplication

Polymerase Chain Reaction (PCR)
 In this reaction, a small fragment of
deoxyribonucleic acid (DNA) or gene can be
rapidly cloned, or duplicated, to produce multiple
DNA copies. It requires:-
 Primers- small chemically synthesised
oligonucleotides that are complementary to the
regions of DNA.
 Taq Polymarase - Thermostable DNA
Polymerase isolated from Thermus aquaticus, a
heat-loving bacterium found in the hot springs of
Yellowstone National Park. It remain active during
the high temperature.
 Nucleotide Bases
Each cycle of PCR consists of three
phases
1. Denaturation: The DNA is heated to
cause its two linked strands to separate.
2. Annealing: The temperature of the
mixture is lowered to allow primers—starter
pieces of DNA—to bind to the separated
DNA.
3. Extension (Polymerization) : The Taq
polymerase enzyme extends the primer using
the nucleotides and copy the DNA rapidly.
 One complete PCR cycle takes less than two
minutes to complete.
 Theoretically, the PCR cycle can be repeated
indefinitely, but the polymerase, nucleotides,
and primers are usually renewed after about
30 cycles. .
 Thirty PCR cycles can produce 1 billion DNA
copies in less than three hours.
 Applications of Recombinant DNA
Technology
1. To understand molecular events in biological processes such
as cell differentiation and aging.
2. It can be used to make precise gene maps.
3. Useful chemical compounds can be produced.
Example: injectable hepatitis B vaccine.
4. In the diagnosis of diseases.
Example:
a. Identification of food poisoning by Salmonella,
b. Hepatitis virus
c. HIV
5. Testing the DNA of parents who are carriers for genetic
disorders can be done and other chances of producing an
afflicted child can be predicted.