DAV INSTITUTIONS

ODISHA ZONE- 1
SUBJECT -- BIOLOGY , CLASS -- XII

BIOTECHNOLOGY: Principles and Processes

{Part- 1}
Prepared By:
Ms.SUREKHA PATRA
PGT BIOLOGY,
DAV PUBLIC SCHOOL,
CDA,CUTTACK.

Work is Worship.
 BIOLOGY
Text Book For Class XII

For reference to the NCERT pdf please visit the

link below:
https://ncert.nic.in/ncerts/l/lebo111.pdf

Work is Worship.
 LEARNING OBJECTIVES
On reading this chapter the students will be able to:
 list various tools used in recombinant DNA technology.
 know about different techniques to alter the chemistry of
genetic material(DNA & RNA).
 understand the action of restriction enzymes on DNA
molecules.
 conceptualise the technique of Gel electrophoresis through
animation.
 draw the structure of pBR322:the most commonly used vector
for rDNA technology.
 isolate DNA from the cell by simple process.
 analyze the role of Taq polymerase in PCR .
 develop clear cut idea about bioreactors and down stream
processing
Work is Worship.
 INTRODUCTION :

 The term Biotechnology was given by Karl Ereky (1919).

 It is the manipulation of living organisms or parts of
living organisms to make products useful to humans .
 It deals with manipulation of genes of organisms to
alter their behavior, characteristics to produce specific
products.
 According to European Federation of Biotechnology(EFB),
Biotechnology is the integration of natural science and
organisms,cells,parts thereof,and molecular analogues for
products and services.
Work is Worship.
 Animal
Bioinformatics Genomics /
Husbandry proteomics

Energy and
environment Plant agriculture
management crop improvement
Biotechnology
Genetic
Food Innovations
engineering

Fermentation Health
technology
Diagnostics pharmaceuticals

Work is Worship.
Biotechnology is studied in two
phases

Traditional (old)
biotechnology

Modern (new)
biotechnology

Work is Worship.
Traditional Biotechnology :
 It includes the process that are
based on the natural capacities of
micro-organisms .
 Curd , vinegar , ghee, wine , and
beer ,idli , dosa , paneer and some
other food have been produced
using traditional biotechnology .

Modern Biotechnology :
New and useful traits in crop varities and animal
breeds are created with the help of genetic
engineering
For example : in vitro fertilization leading to a ‘ test
tube baby ‘
Large scale production of desired quality product
from microorganisms (bio- reaction)

Work is Worship.
 Principles of Biotechnology
The two core techniques that developed modern
Biotechnology are
Genetic engineering : The technique of altering the
chemistry of DNA(genetic material) and introduce it
into host organism to change its phenotype.
Sterilisation Methods : Maintenance of sterile
atmosphere to enable growth of only the desired cells
in large quantities for the manufacture of
biotechnological products like antibiotics, vaccines
enzymes etc.

Work is Worship.
Techniques Of Genetic Engineering

Creation of Recombinant DNA

Gene transfer into host organism

Gene Cloning

Work is Worship.
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.

Work is Worship.
 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.

Work is Worship.
• 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.
Work is Worship.
Molecular Tools Of Genetic Engineering :
Key tools required for the recombinant DNA technology
are :

Restriction enzymes

Polymerase enzymes

Ligases

Vectors

Host organisms / cell

Work is Worship.
 RESTRICTION ENZYMES :
 Restriction Enzymes (RE) are called “ molecular
scissors “ are responsible for cutting DNA at specific
sites.
 Restriction enzymes belong to a class of enzymes EcoR I
called nucleases and are of two type
i) Exonucleases – degrade DNA from the
terminal ends .
ii) Endonucleases – act on the integral
phosphodiester bonds .
 These are the bacterial enzymes that can cut / split
DNA (from any source ) .
 They were first discovered in E. coli restricting the
replication of bacteriophages , by cutting the viral DNA
( the host E.coli DNA is protected from cleavage by
addition of methyl groups ) .
 The first restriction endonucleases , HindII , was
isolated by Smith , Wilcox and Kelley (1968) .
Work is Worship.
 NOMENCLATURE :
Restriction Endonucleases are named by a standard procedure,with particular reference
to the bacteria from which they are isolated .

E co R I
First endonuclease
Ry13
coli
Escherichia
 First letter (in italics) – indicates the genus name .
 Next two letters (in italics) – the species name .
 Next letter – strain of the organism .
 Roman numeral indicating the order of discovery .
 HindII -
H ( Haemophilus ),in(influenza ), d ( strain Rd ) , II ( second endonucleases)

Work is Worship.
 TYPES OF RESTRICTION ENZYMES :

TYPES SALIENT FEATURES

A single enzyme with 3 subunits for recognition ,
I cleavage and methylation . It can cleave up to
1000bp from recognition site.

Two different enzymes either to cleave or modify

II the recognition sequence . Cleavage site is the same
or close to recognition site.

A single enzyme with 2 subunits for recognition and

III cleavage . Cleavage site is 24-26 bp from recognition
site.

Two different enzymes , cleavage site is up to 20 bp

IIs from recognition site .

 The recognition sequence of endonucleases are palindromic

5’ ----- G A A T T C ------3’
3’ ------ C T T A A G -----5’

Work is Worship.
MECHANISM OF ACTION OF ENDONUCLEASE :

Work is Worship.
 STEPS OF ACTION OF ENDONUCLEASES

On finding the palindrome, the endonuclease binds to the DNA.

Same restriction endonuclease is required to cut both the DNA fragments have
same kind of sticky ends.

ER cuts the opposite strands of DNA in the sugar – phosphate backbone (little away
from the center of the palindrome site between same bases on both strands )

Single stranded overhanging stretches at the end of each strand called – sticky end .

Sticky ends are complementary to each other .

Sticky ends facilitate the action of enzyme DNA ligase ( forming H- bond with
complementary strands) end -to - end . Work is Worship.
SO NOW LETS TAKE A SHORT RECAP BY
WATCHING A QUICK INTERACTIVE
VIDEO

https://www.youtube.com/watch?v=GJrAsW41a64&t=10s

Work is Worship.
 Agarose gel Electrophoresis

 Electrophoresis through agarose gels is the standard method

for the separation , identification and purification of DNA
and RNA fragments ranging in size from few hundred to
20Kb .

 Polyacrylamide gel electrophoresis is also used for the

same purpose , but is preferred for small DNA fragments .

 Agarose gel electrophoresis simple and rapid and capable of

resolving DNA fragments that cannot separated by density
gradient centrifugation .
 Location of DNA within the gel can be determined directly
by staining with low concentrations of fluorescent ethidium
bromide dye under UV light .

 DNA recovered from gel and used for a variety of cloning

purposes .
Work is Worship.
SCHEMATIC REPRESENTATION OF GEL ELECTROPHORESIS ACTION
Work is Worship.
AND NOW LETS LEARN THIS GEL
ELECTROPHORESIS THROUGH A
QUICK INTERACTIVE VIDEO

https://www.youtube.com/watch?v=4OJAzQsZnbo&feature=youtu.be

Work is Worship.
 PROCEDURE
 Agarose gels are cast by melting the agarose (linear polymer
of D-galactose and 3,6-anhydro L- galactose ) extracted from
seaweed in the presence of desired buffer .
 Melted solution poured into a mould and can be made into
different size and porosities and gel allowed to harden .
 Electricity is applied across the gel , DNA which is negatively
charged at changed pH migrate towards anode .
 The rate of migration depends on
1.Molecular size of DNA
2.Agarose concentration
3.Composition of DNA
4.Composition of electrophoresis buffer (in absence
of ionic buffer electrical conductivity is minimal and
DNA migrates very slowly )
 Purified DNA fragments are used to form recombinant DNA
which can be joined with cloning vectors .

Work is Worship.
 CLONING VECTORS
 Vectors are the DNA molecules , which can carry a foreign
DNA fragment to be cloned . They are self replicating in an
appropriate host cell .
Characteristics of an ideal vector :
 Vector should be small in size , with a single restriction
endonuclease site , an origin of replication and 1-2
genetic markers .
 Vectors may be :
Plasmids
Bacteriophage

Work is Worship.
A) Plasmids : plasmids are extrachromosomal , double- stranded ,
circular , self-replicating DNA molecules
 Almost all the bacteria have plasmids containing a low copy number
(1-4 per cells ) or high copy number (10-100 per cell ) .
 Plasmids contribute about 0.5 to 5.0% of the total DNA of bacteria .
Types of plasmid :
1. a) Conjugative – they carry set of transfer genes (tra genes )
facilitates bacterial conjugation
b) Non-conjugative – they don’t have tra genes
2. Another classification is based on the copy number
a)Stringent plasmid : present in a limited number (1-2 per cell)
.
b)Relaxed plasmid : occur in large number in cell .
3. F-plasmids : possess genes foe their won transfer from one cell
to another .
R- plasmids : carry genes resistance to antibiotics

Work is Worship.
 NOMENCLATURE OF PLASMIDS

 To designate plasmid by a lower case p followed by the first letter (s) of

researchers names and the numerical number given by the worker

pBR322
plasmid

Bolivar and Rodriguez

Plasmid designated as 322

 Some plasmids are given name of the place where they are discovered .
 E.g., pUC – plasmid from university of California .

Work is Worship.
Cloning vector
Work is Worship.
ART INTEGRATION

https://youtu.be/LtRrvKAhVY8

Work is Worship.
 BACTERIOPHAGE
 Bacteriophages or simply
phages are virus that replicate
within the bacteria .
 Phage vectors can accept short
fragments of foreign DNA into
their genomes .
 The advantage with phases is
that they can take up larger DNA
segments than plasmids .
 Bacteriophage lambda ( phase
λ) , virus of E. coli , has been
more thoroughly studied and
developed as vector .
 importance of of phase λ ,
about 50% of phase DNA
necessary for its multiplication
and other functions .
Work is Worship.
An ideal vector should have following features to facilitate
cloning :
Origin of replication site :
 Ori-site is the DNA sequence that is responsible for initiating
replication .
 Ant DNA can linked to this sequence can replicate within the host cell .
 Ori also control copy numbers of the linked DNA .
Selectable marker :
 It helps to select the host cell which contain the vector
(transformation ) and eliminate the non-transformants .
 Transformation is defined as the procedure by which a piece of DNA is
introduce into a bacterial host .
 Gene encoding resistance to antibiotics like ampicillin ,
chloramphenicol , tetracycline , kanamycin are useful selectable
markers for E. coli as the normal E. coli do not carry resistance
against these antibiotics .
Work is Worship.
Cloning site :
 To link the alien DNA vector requires more
restriction sites to generate high copy number are
called multiple cloning site or poly linker .
Vectors for cloning genes in plants and animals :
 In plants , tumor inducing plasmid (Ti) of
Agrobacterium tumifaciens is used as cloning vector .
It deliver TDNA in the Ti plasmid which transfer
normal plant into cells to produce chemical against
pathogen .
 Retrovirus , adenovirus , papillomavirus are also now
used as cloning vector n animals .

Work is Worship.
Work is Worship.
Selection Of Recombinants :
Inactivation of antibiotic resistance genes :
 If a foreign DNA ligated at the BamHI site of the
tertracycline resistance gene in the vector pBR322 , the
recombinant plasmid loses the tetracycline resistance due
to insertion of the foreign gene.
 It can be selected from the non-recombinant one by plating
recombinant on ampicillin and then tetracycline containing
medium .
 The recombinant can grow in ampicillin containing medium
but not in tetracycline medium but the non- recombinant
can grow on both the media.

Work is Worship.
Lets learn Insertional Inactivation through this
short videO

https://www.youtube.com/watch?v=eA9Kw4d_O9Y&list=WL&index=1&t=2s

Work is Worship.
Insertional inactivation :
Recombinants and non-recombinants are
differentiated on the basis of colour production
in the presence of chromogenic substance.
Foreign DNA is inserted within the coding sequence
of an enzyme ß- galactosidase , which results in
inactivation of enzyme.
Colonies with inserted plasmid showing no
coloration , while non-inserted plasmid form blue
color colonies

Work is Worship.
And now lets learn the Blue-White Screening
Technology through this interactive video

https://www.youtube.com/watch?v=Y7gxELssMRw&feature=youtu.be

Work is Worship.
THANK YOU

Work is Worship.