BIOTECHNOLOGY–PRINCIPLES AND PROCESS

 Biotechnology deals with the techniques of using organisms or enzymes

to produce products useful to humans.
 It will include all microbe mediated process (e.g. making vine, curd etc.),
invitro fertilisation leading to ‘test tube’ baby synthesizing a gene etc.
 The term biotechnology was proposed by a Hungarian Engineer, Karl
Ereky (1917)
• Based on the development in biotechnology, it may be of 2 types:-Old
Biotechnology and Modern Biotechnology.

Old Biotechnology
Old Biotechnology deals with the techniques in biotechnology based on the
natural capabilities of microorganisms.
E.g.- Preparation of wine, beer and other alcoholic beverages, vinegar ,cheese
, curd, Idli, dosa and some other foods.
All are microbe mediated processes.
• Micro-Organisms are being used for making curd, wine, vinegar, bread
etc. for thousands of years.

2. Modern Biotechnology
• Modern Biotechnology deals with the techniques in biotechnology which
use genetically modified Organisms, (GMO s) to achieve the products of
traditional biotechnology on a large scale.
• The important techniques included under modern biotechnology are the
following
1. Techniques like invitro fertilization leading to a test tube baby.
2. Synthesising a gene and using it.
3. Developing a DNA vaccine.
4. Correcting a defective gene.
In 1978, the European Federation of Biotechnology (EFB) has defined
biotechnology as “The integration of Natural Science and Organisms,
cells, parts thereof and molecular analogues for products and services”.

Principles of Modern Biotechnology

Modern biotechnology is genetic engineering. Its core techniques are:
(i) Genetic Engineering – Technique of cutting and pasting a desired DNA
or RNA to a vector and then introducing to host organism. The resultant
organism called transgenic organism.
• Paul Berg is considered as Father of Genetic Engineering. He was
awarded the Nobel Prize in 1980.
(ii) Biochemical Engineering process – Technique of maintenance of sterile
(microbial contamination free) medium for large scale growth of desired
transgenic organism for manufacture of biotech products like antibodies,
vaccines, enzymes etc.

Conceptual development of the Principles of Genetic Engineering

• During Sexual reproduction, different genetic makes ups (from male and
female) combine to form a new genetic set up.
• Organisms reproduce either by asexual or sexual methods.
• Variations are also created during sexual reproduction.
• Some of the variations are beneficial to the organism and also to the
Population.
• Asexual reproduction preserves genetic set up. Variations are not created
during asexual reproduction .So sexual reproduction is more advantageous
than asexual reproduction.
• Sexual reproduction produce variations and asexual reproduction
preserves these variations.
• Sexual reproduction is advantageous since it permit variations and
recombination.
• Traditional hybridization methods are commonly used in Plant breeding
and animal breeding.
• In these methods, the undesirable genes are multiplied along with the
desirable genes. The undesired genes are very difficult to remove .This
drawback has been overcome with the development of genetic
engineering.
• Genetic Engineering deals with the Manipulation of genes. It includes the
artificial synthesis, modification, removal, addition and repair of the
DNA (genetic material) to get a desired and useful phenotype.
 • Genetic Engineering is often referred to as recombinant DNA technology.
• Microorganisms are commonly used to study the structure and expression
of genes. This is because it is easy to isolate mutants with altered traits.
• In genetic engineering technique, the DNA or genes of different
organisms are joined to produce hybrid DNA.
• The hybrid DNA is called recombinant DNA or rDNA.
• The subject of genetic engineering or recombinant DNA technology
involves the transfer of specific genes from one organism to another
through the use of appropriate enzymes.
It actually involves manipulation of the two DNA fragments (genes)
involved, thus the term “genetic engineering”
has come in to being.
• In 1972, Stanley Cohen , Standford University USA and Hebert Boyer,
University of California USA invented the process of genetic engineering
by combining a piece of foreign DNA containing a gene from a bacterium
with a bacteria ([Link]) Plasmid using the enzyme restriction
endonuclease.

The technique of genetic engineering has 3 steps.

1. Creation of recombinant DNA.
 2. Use of gene cloning.
3. Gene Transfer.
• Genetic Engineering permits us to isolate and introduce only one set or a
set of desirable genes without introducing undesirable genes in to the target
organism.
Construction of first Artificial Recombinant DNA molecule
• The construction of the first recombinant DNA emerged from the
Possibility of linking a gene encoding antibiotic resistance with a native
plasmid of Salmonella typhimurium.
• Plasmid is self-replicating extra –chromosomal circular DNA present in
some bacteria.
• Stanley Cohen and Hebert Boyer constructed first recombinant DNA in
1972, by isolating the antibiotic resistance gene by cutting out a piece of
DNA from a plasmid which was responsible for giving antibiotic
resistance.

• The cutting of DNA at specific locations became possible with the

discovery of restriction enzymes popularly called “Molecular Scissors”.
• The cut piece of DNA was then linked with the plasmid DNA .These
plasmid DNA act as vectors to transfer the piece of DNA attached to it.
(Female Anopheles mosquito acts as an insect vector to transfer the Malarial
parasite in to human body. In the same way, a plasmid can be used as vector to
transfer an alien of piece of DNA (foreign DNA) in to the host organism)
• The linking of antibiotic resistance gene with the help of enzyme DNA
ligase.
DNA ligase acts on cut DNA molecules and join their ends. This makes a
new combination of circular autonomously replicating (self-replicating)
DNA, created invitro .It is known as recombinant DNA (rDNA).
 • When this recombinant DNA is transferred in to [Link], a bacterium
closely related to Salmonella, it could replicate using the new host’s DNA
polymerase enzyme and make Multiple copies.
The making of multiple copies of antibiotic resistance gene in [Link] was
termed as cloning of antibiotic resistance gene in [Link].

From the above discussion, it is quite clear that there are 3 basic steps in
creating genetically modified Organisms (GMOs).
1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA in to the host.
3. Maintenance of introduced DNA in the host and transfer of DNA in to its
progeny.
ANDi (a backward abbreviation for inserted DNA) with glistening nails –
World’s first genetically altered primates by inserting a jelly fish gene (gene for
glistening) in to the embryo of a rhesus monkey.
• At what stage of meiosis is a recombinant DNA made naturally?
During meiosis I, recombinant DNA is formed by crossing over in
pachytene stage of Prophase I.
TOOLS OF r-DNA TECHNOLOGY
The following key tools are necessary to accomplish the r-DNA technology.
They are: 1. Restriction enzymes
2. Polymerase enzymes
3. Ligases
4. Vectors
5. Host Organisms.
1. Restriction enzymes
• Enzymes which cut DNA at specific sites (specific points) in to fragments
containing identifiable genes, are called restriction enzymes.
 • The restriction enzymes also called “Biological Scissors” or “Molecular
Scissors” or “chemical knives” or “Chemical Scalpels”.
• Hundreds of restriction enzymes with different sequence specificities
have been isolated from many bacteria, and are commercially available.
• The important restriction enzymes are the following.
1. Restriction Endonucleases
2. Hind III
3. Hind II
4. Bam HI
• The commonly used restriction enzyme is restriction endonuclease.
• The restriction endonuclease cleaves (cuts) double stranded DNA (ds
DNA) at specific sites in to DNA fragments.
Discovery Restriction Enzymes.
• In 1963, Steward Linn and Werner Arber discovered that Escherichia Coli
is able to protect itself from the attack of bacteriophages (Bacteriophage,
any of a group of viruses that infect bacteria) with the help of a defence
mechanism called Restriction Modification System.
This system has 2 components
1. Restriction Enzymes
2. Modification Enzymes.
3. Restriction Enzymes
 This enzyme identifies the introduced foreign DNA (alien DNA)
and cuts it in to pieces.
 It restricts the propagation of foreign DNA.
2. Modification Enzymes
It adds a methyl group to one or two bases with in the sequence identified by
restriction enzyme.
If a base in DNA is modified due to addition of methyl group, restriction
enzyme cannot identify and cleave (Cut) the DNA.
By this method, bacteria are able to protect their own chromosomal DNA from
cleavage by Restriction enzymes. Thus, bacteria bear sets of Restriction
Endonucleases and corresponding methylases.
Types of Enzymes in Organisms
Enzymes are biological catalyst capable of promoting chemical reactions
in the living system.
In 1961, International Union of Biochemistry (IUB) classified enzymes in to
6 large classes (group).They are given below.
 1. Oxido reductase
2. Transferase
3. Hydrolase
4. Lyase
5. Isomerase
6. Ligases.
• Of these 6 groups, hydrolases are enzymes which break down complex
molecules in to smaller ones.
• E.g. Carbohydrase, Lipases, Proteinase , Nuclease.
Enzymes which breakdown nucleic acids are called Nucleases.
Nucleases are of 2 types- Exonucleases and Endonucleases.
Exonuclease
The nuclease enzyme which removes nucleotides from the ends of the DNA,
is called exonuclease.
Endonuclease
The nuclease enzyme which cuts at specific sites (points) with in the DNA, is
called Endonuclease.
E.g.:- Restriction Enzymes.
• The important restriction enzymes are restriction Endonucleases (REN).
• A group of endonucleases which cut at specific sites (particular points)
with in the DNA, is called restriction endonucleases (REN). The specific
site with in a specific base sequence of DNA is known as the recognition
sequence. It consists of 4 and 8 base pairs (4 to 8 nucleotide).The
recognition sites differ for different restriction enzymes.
• Now more than 900 restriction enzymes that have been isolated from over
230 strains of bacteria each of which recognize different recognition
sequences .
• Restriction Endonucleases serve as the tools for cutting DNA molecules
at pre -determined sites, which are the basic requirement for gene cloning
or recombinant DNA technology.
Types of Restriction Endonuclease
The restriction endonucleases are classified in to 3 groups
1. Type I Restriction Endonuclease.
2. Type II Restriction Endonuclease.
3. Type III Restriction Endonuclease.
Out of these, only Type II restriction endonucleases are used in
recombinant DNA technology, because they can be used invitro to recognize
and cut with in specific DNA sequence with 4 to 8 base pair.
• For example Hind II always cut DNA molecules at a particular point
(sites) by recognizing a specific sequence of 6 base pair. This specific
base sequence is known as the recognition sequence for Hind II.
 • The first restriction endonuclease discovered was Hind II (from
Haemophilus influenza) by smith (1968).It was found to cut DNA at
particular points having specific sequence of base pairs.
• The restriction endonuclease was first isolated by Nathans and Smith
were awarded Nobel Prize in (1978) for the discovery of restriction
endonuclease (EcoRI)
• Restriction endonucleases are present in Bacteria .They protect bacteria
from viral attack by disintegrating viral DNA without harming bacterial
genome .(Eukaryotic cells do not have restriction endonuclease)
• Nomenclature of Restriction Enzymes.
• Naming of restriction enzymes is usually done by following way.
• First letter of the name is taken from the genus name (in italics) of the
prokaryotic cell (e.g. bacteria) from which they been isolated.
• Second two letters (also in ltalics) are taken from the first 2 letters of the
name of the species.
• Fourth letter is taken from the first letter of name of the strain.
• Last letter is a Roman number indicating the order in which the enzymes
were isolated from that strain of bacteria (if an organism forms many
enzymes, they are identified by sequential Roman numericals).
• For example, EcoRI comes from Escherichia coli RY13.
• E –Escherichia (genus name of prokaryotic cell from which EcoRI is
isolated)
• Co- co stands for coli (species name).
• R-R stands for RY 13 (name of the strain of the bacterium)
• I-It was the first endonuclease (I) isolated from that strain of bacteria.
Style of DNA cleavage
The restriction enzymes cut DNA molecule by cleavage. This action
occurs in 2 styles .They are sticky-end style and blunt-end style.
1. Sticky-end style
• In this style, the restriction enzymes make single- strand cut. Thus, the
cut ends of the DNA fragments will be protruded out (single –stranded
hanging structures).The ends of each DNA fragments are called sticky
ends or cohesive ends or staggered ends.
• This stickiness of the ends facilitates enzyme DNA ligase in joining DNA
fragments having the same kind of sticky [Link]: Eco RI (from [Link]),
Bam HI (from Bacillus amyloliquefaciens), Hind III (from
Haemophilus influenza), etc.
• EcoRI cleaves both strands at different points (different recognition sites)
 2. Blunt –end style
• In this style, the restriction enzymes make cut across both strands of DNA
at the same position of the recognition site. Thus, cut ends of DNA
fragments are in blunt appearance. The ends of each DNA fragments are
called blunt ends or flush ends.
E.g. Sma I (from serratia marcescens -rod shaped bacterium)
Sma I recognizes the 6 nucleotide Palindromic sequence and cleaves at
the both the ends.
• In the case of blunt –end DNA fragments, the linkers are to be used.
• Linkers are short, chemically- synthesized double –stranded DNA .
Linkers can be ligated to the blunt ends of the DNA fragments and then
treated with restriction enzyme to produce sticky –ends.
Action of Restriction Endonuclease
• The recognition sequence with recognition site within the DNA is called
Palindromic nucleotide sequence.
• The palindrome is a group of letters that reads the same forward and
backwards. For example MALAYALAM.
• The palindrome in DNA is a sequence of base pairs that reads same on
the 2 strands when orientation of reading is kept the same.
• For example, the following sequences read the same on the 2 strands in 5’
3’ direction. This is also true when we read in the 3’ 5’ direction
 • Each restriction endonuclease functions by inspecting the length of a
DNA sequence.
• Once it finds its specific recognition sequence in DNA, and it will bind
to the DNA and cut each of the 2 strands of the double helix of DNA at
specific points in their sugar- Phosphate backbones.
• Each restriction endonuclease recognizes a specific palindromic
nucleotide sequence in the DNA.
• Restriction enzymes cut strands of DNA a little away from the centre of
the palindrome sites, but between the same 2 bases on the opposite
strands.
• As a result, the DNA fragments produced by the cut of these enzymes
have sticky ends.
When cut by the same restriction enzymes, the resultant DNA fragments
have the same kind of ‘sticky ends’. These can be joined together end to
end using DNA ligase.

II. Polymerase Enzymes

Enzymes which help in the synthesis of molecules are called polymerase
enzymes. E.g. DNA polymerase.
DNA polymerase
 It helps in the synthesis of complementary DNA (cDNA) strand to an
existing DNA template. The enzyme DNA polymerase was first discovered by
Korenberg. In 1956, he identified 3 types of DNA polymerases in [Link].
1. DNA polymerase I (DNA pol I)
2. DNA polymerase II (DNA pol II)
3. DNA polymerase III (DNA pol III).
They have almost similar catalytic activity, however, DNA polymerase III is
more active than the other two.
III. LIGASES
Enzymes which are used to join the molecules, are called ligases.
E.g. DNA ligase.
DNA ligase
• It is an enzyme which is used to join the DNA fragments. So DNA ligase
is called “Chemical Suture” or “molecular glue”. It is obtained from
[Link].
• In rDNA technology, the most commonly used joining enzyme is T
4
4
DNA ligase. ( the phage encoded T DNA ligase is produced during
4
infection of [Link] by bacteriophage T ).

Separation and isolation of DNA fragments

• When DNA is treated with restriction endonuclease, it breaks up in to
many DNA fragments of variable lengths. These DNA fragments can be
separated by a technique called gel electrophoresis.

Electrophoresis
• Is a technique of separation of charged macromolecules under the
influence of an electric field with cathode (-) and anode (+) and a matrix
(medium).
• The charged molecules migrate in the electrode bearing the opposite
charge through the matrix.
• Commonly used matrix may be polyacrylamide, in polyacrylamide gel
electrophoresis (PAGE) or Agarose (in Agarose gel electrophoresis or
AGE).
• Now a days, Agarose is the most commonly used matrix.
• Agarose is a natural polymer extracted from sea weeds.
• Electrophoresis was first developed by [Link] and Longsworth in
1937.

• The DNA fragments are placed over the cathode end.

• The electric field is activated. DNA Fragments are negatively charged
molecules. So, DNA fragments move towards the positively charged
anode end.
• Agarose has a sieving effect.
• The DNA fragments are separated (resolved) according to their size
through sieving effect provided by the Agarose gel (through the pores of
Agarose gel).
• Larger DNA fragments move slowly while the smaller DNA fragments
move fast and reach the farther end.
• Pure DNA fragments cannot be seen in visible light and without staining.
• DNA fragments are stained with ethidium bromide, and exposing to UV
radiation.
• DNA fragments appear as bright orange- colored bands in ethidium
bromide stained gel exposed to UV light.
• The separated bands of DNA are cut out from the Agarose gel and
extracted out from the gel piece. This process is called elution (removal
of adsorbent).
• The purified DNA fragments are then used in constructing recombinant
DNA s by attaching (joining) them to cloning vectors.
• DNA library or gene library can be constructed by this method. The
genes can be identified through their products.
 • The process of making identical copy or copies of a single parent is called
cloning.
Vectors (Cloning Vectors)
The vehicles which are used to transfer a gene (DNA fragments)
from one cell to another are called vectors. Vectors
Are also called cloning vectors or cloning vehicles.
The commonly used cloning vectors are the following
1. Plasmid vectors
2. Phage vectors
3. Cosmid vectors
4. Shuttle vectors
5. Transposons.

1. Plasmid Vectors
• The plasmid used as a cloning vector is called plasmid vector.
• Plasmid is a circular, double –stranded, extra-chromosomal DNA
present in bacterial cell.
• It is self- replicating.
• Commonly used naturally –occurring plasmid vectors are plasmid
vectors are Plasmids of Escherichia coli (E .coli) and
Agrobacterium tumifaciens.
• The naturally –occurring plasmid in [Link] is called ColE1 and in
Agrobacterium tumifaciens is called Ti Plasmid.
• Naturally –occurring plasmids may not possess all the properties of a
suitable cloning vector. Therefore ,it may be reconstructed by inserting
certain other genes.

• The important commercial artificially –reconstructed plasmid from the

natural plasmid of [Link] are pBR322, pUC-18,etc.
1. pBR322
It is an artificially –reconstructed plasmid vector from the plasmid of
[Link] (Col E1).During its reconstruction, 3 genes are added to Col E1.
1. ori – It is the gene for relaxed replication.
R
2. tet -It is the gene for tetracycline.
R
3. amp – It is the gene for ampicillin

R , R
tet amp -are the genes for resistance against antibiotics.
The pBR322 was prepared by Bolivar and Rodriguez “p” refers to
plasmid, “B” to Bolivar and “R” to Rodriguez.
 2. pUC-18
It is an artificially -reconstructed plasmid from the plasmid of [Link] (Col
E ).During its reconstruction, 2 genes are added to Col E
1 1.
1. ori- it is the gene for relaxed replication.
R-
2. amp It is the gene for ampicillin.
R
amp is the gene for resistance against antibiotics.
R,
In addition to ori and amp ,a multiple cloning site called lacZ is
also seen. pUC -18 can form up to 500 copies of host cells.

3. Phage vector
 It is bacteriophage used as cloning vector. The DNA of lamda phage is
commonly called as phage vector. It is double Stranded and linear.
4. Cosmid vector
It is an artificially –prepared vector by inserting a ligating DNA sequence
(called cos site) from lambda phage in to a plasmid.
5. Shuttle vector
It is a specially designed vector capable of replication in both prokaryotic
(e.g. Bacteria) and eukaryotic (e.g. Yeast) host cells.
6. Transposons
DNA sequence that can transposes (jumps) from one location to
another , is called transposon (jumping gene).
Features of a cloning Vector
To act as vector, DNA molecule should bear certain characteristics such
as origin of replication, selectable marker, cloning sites and vectors for cloning
genes.
1. Origin of Replication (ori )
The gene (a DNA sequence) responsible for starting of replication is
called ori or origin of replication.
Characters of ori
1. Ori initiates the replication.
2. Any piece of DNA when linked to ori can be made to replicate within the
host cells.
3. Ori possesses replication control of the linked [Link]: when many copies
of the target DNA (foreign DNA) are required .it is introduced (cloned) in to a
vector. This foreign DNA will replicate many times and form several copies.
Therefore, the selected plasmid should have an origin of replication (Ori). That
supports high copy number.
• Prokaryotic DNA has normally a single origin of replication (ori).
• Eukaryotic DNA have more than one origin of replication (ori) .
2. Selectable Marker
The gene which helps in identifying and eliminating non- transformants
and selectively permitting the growth of the transformants, is called selectable
marker.
A bacterium which is modified through a foreign gene (a piece of DNA) is
called transformant.
• The process through which a foreign gene (a piece of DNA) is introduced
in to a host bacterium is called transformation. The useful selectable
markers for [Link] are the following.
R -
[Link] It is the gene for resistance against an antibiotic, tetracycline.

R
2. amp -It is the gene for resistance against an antibiotic, ampicillin.
 Normally, naturally –occurring [Link] cells do not carry resistance against
any of these antibiotics.

• A vector requires a selectable marker gene. E.g.: pBR 322 has both genes
R R
for ampicillin resistance (amp ) and tetracycline resistance (tet ) in
addition to ori.
R
• pUC -18 has only ampicillin resistance gene (amp ) in addition to ori.
3. Cloning sites

• The recognition sites for the activity of commonly used restriction

enzymes in order to link the alien DNA (foreign DNA) ,are called
cloning sites.
• The vector must have single cloning sites. Occurrence of more than one
cloning sites will cause DNA fragmentation.
• Gene cloning will become more complicated due to formation of many
DNA fragments.
• The ligation of foreign DNA is carried out at a restriction site (cloning
site) present in one of the 2 antibiotic resistance [Link]: in the vector
pBR 322, alien DNA is ligated (jointed) in the area of Bam HI site of
tetracycline resistance gene.
• Such recombinant plasmid will loose tetracycline resistance due to
insertion of foreign DNA (alien DNA).rop is a gene which codes for the
proteins involved in the replication of the plasmid

[Link] cloning vector pBR322 showing restriction sites (Hind III, Eco RI,
Bam HI, Sal I, Pvu II, Pst I, Cla I), Ori and antibiotic resistance genes
R R
(amp and tet ) and rop codes for the proteins involved in the replication
of the plasmid.
Vectors for cloning genes in Plants and Animals
Gene are transferred from bacterium or virus to eukaryotic cells.
There are various procedures of transferring genes in to plants and animals from
bacteria and viruses. The transferred genes force the transformed cell to work at
their will.
E.g. agrobacterium – mediated gene transfer.
Gene Transfer
The process of gene transfer through vectors is called vector-through
gene [Link] is indirect gene transfer .The commonly used cloning vectors
are plasmid vectors, phage vectors, cosmid vectors, shuttle vectors, transposons,
etc.
[Link] and Agrobacterium tumifaciens are the commonly used
plasmid vectors. The artificial chromosome used for gene transfer from bacteria
is called BAC ( Bacterial artificial chromosomes) and from Yeast is called YAC
(yeast artificial chromosome).
Natural genetic “Engineer”
• Agrobacterium tumifaciens is a soil bacterium which is pathogenic to
plants.
• It causes crown gall (plant tumour) disease in dicot plants such as tomato,
potato, tobacco etc.
• It can transfer a part of its plasmid DNA in to the plant cells as it infects
the host plants.
• It is the most important tool in genetic engineering in plants.
• The plasmid of Agrobacterium is called Ti-plasmid (tumour –inducing
plasmid).Ti plasmid has an important transferable part called T-DNA.
• T-DNA contains many genes, of which the gene “tra” helps in its transfer.

• The bacterium also contains a tumour –causing gene. Agrobacteruim

tumifaciens can pass on its tumour-causing gene in to the genome of the
infected plant (host plant) .
• As a result, galls (tumour) are formed on the host plant.
 • Therefore, Agrobacterium tumifaciens is known as the “natural genetic
engineer” of plants, as here, the gene transfer is happening in nature
without human interference.
• During gene cloning technique, the desired gene is to be inserted with T-
DNA. So, when the hybrid DNA enters in to a plant cell, the T-DNA
along with the desired gene is separated and integrated within the DNA in
the nucleus of the plant cell and thus the plant cell (host cell) gets
genetically modified.
• Ti –plasmid based vectors have been most commonly used for the genetic
transformation of many dicot plants such as tomato, potato, sunflower ,
cotton etc., and of monocot plants such as cereals like rice, wheat, and
corn .
• The gene transfer through Agrobacterium is called Agrobacterium
mediated gene transfer.
• Cauliflower mosaic virus (CaMV) is an important gene cloning vector
used for plants.
Vector less gene Transfer
• The process of transference of a gene directly in to host cells, is called
vector less gene transfer.
• It is the direct gene transfer.
• Recently new methods are developed for vector less gene transfer.
• The important methods are micro-injection, gene gun or biolistics gun,
etc.
Micro injection
• Introduction of a recombinant DNA (foreign gene) in to the nucleus of an
animal cell using micropipettes (glass needles) is called micro-injection.
• In 1993, Jeffey [Link] (Human genome centre ,Michigam
University ,U.S.A ) cured mice suffering with a neuromuscular disease
which is like muscular dystrophy of humans, using micro-injection.
Micro- injection procedure for injecting recombinant DNA in to
the nucleus of an animal cell

b. Gene gun (Biolistics)

• It is a type of vector less gene transfer.
• In this method, micro –particles of gold or tungsten coated with foreign
DNA are bombarded with high velocity in to the plant cells, using helium
pressure particle gun device.
• This method was first developed for plants by Stanford (Cornell
University, USA) in 1987.
• It is also used to insert genes in to animal cells that promote tissue repair
near wounds.

C. Disarmed pathogen vector

• It is a type of vector gene transfer.
• This method uses “disarmed pathogen” (transformed pathogen incapable
of causing disease) vectors.
• This pathogen with recombinant DNA vector, when allowed to infect the
cell, transfer the recombinant DNA in to the host cell.
Host Organism
• Host cells or host organisms are required for propagation of DNA
molecules.
Competent Host
• The ability to pick up genes and plasmids from outside, is called
competence.
• The cell which is capable of taking up alien DNA (foreign DNA) is called
competent host. A competent host is essential for transformation with
recombinant DNA.
• Host cells like [Link], yeast , plant cell, and animal cell are being used.
• Most popular and extensively used bacterium is [Link].
• DNA is a hydrophilic molecule.
• So, it cannot pass through cell membranes.
• However, competence can develop for a short duration towards the end of
growth period.
• Bacterial host cell is made competent to take up plasmid.
• For this, bacteria are treated with specific concentration of a divalent
cation like calcium.
• This enhances (increases) the efficiency of entry of DNA in to bacterium
through pores on cell wall.
• Recombinant DNA can be forced in to such cells by following steps.

1. Incubation of cells with recombinant DNA on ice.

0
2. It is followed by giving them a brief heat shock at 42 C and again putting
back on ice.
This makes the bacterium take up the recombinant DNA .The foreign
DNA passes through cell membrane
and enter the cell.
PROCESSES OF r-DNA TECHNOLOGY
Recombinant DNA (rDNA) technology or genetic engineering is
technically a high precision process .It has several steps in a specific sequence.
The stages in r-DNA technology are given below:
1. Isolation of source DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of a desired DNA fragment.
4. Ligation of the DNA fragment in to a vector.
5. Transferring the recombinant DNA in to the host.
6. Culturing the host cells in a medium at large scale.
 7. Extraction of the desired product.
1. Isolation of source DNA.
• In most of organism, the genetic material is DNA .DNA molecules are
seen inside the cell.
• It has to be obtained in pure form and free from other macromolecules
such as RNA, proteins, polysaccharides, and lipids.
• Therefore, the bacterial cells or plant tissues or animal tissue are treated
with enzymes such as lysozyme (for bacteria), cellulase (for plant tissue)
and chitinase (for fungal cells).
• This will break the cell envelop open and macromolecules like DNA,
RNA, proteins, Polysaccharides, and lipids will be released.
• In eukaryotic cells, DNA is intertwined with basic proteins like histones.
• Proteins can be removed by treating it with the enzyme protease.
• RNA and lipid can be removed by treating it with enzymes, ribonuclease
and lipase respectively.
• Other macromolecules can be removed by treatment with appropriate
enzymes.
• The purified DNA, finally precipitates out after the addition of chilled
ethanol (ethyl alcohol).
• The precipitated DNA appears as a mass of very fine threads in the
suspension.
• The DNA threads can be removed by spooling .It is a process by which
DNA threads wind on a reel (spool = reel for winding yarn).

2. Cutting Of DNA at specific locations

• Purified DNA molecules are subjected to restriction enzymes at optimal
conditions of enzyme activity.
• The progress of restriction enzyme digestion is checked through Agarose
gel electrophoresis.
• DNA is a negatively charged macromolecules .So it moves towards the
anode (positively electrode).
• The vector DNA is also cut with the same restriction enzymes so as to get
complementary sticky ends.
• After cutting the source DNA and the vector DNA with a specific
restriction enzyme, the cut out “gene of interest” (desired gene)
from the source DNA and cut vector DNA having the cut space
are mixed and the enzyme ligase is added.
 • The enzyme ligase joins the desired gene (gene of interest) with the cut
vector DNA to produce a recombinant DNA (r DNA or Hybrid DNA or
chimeric DNA).
• The process of formation of hybrid DNA is called ligation or gene
splicing.
• The vector with the desired gene (DNA fragment) is called recombinant
vector or chimeric vector.
• The commonly used ligase is DNA ligase.
3. Amplification of gene of interest using PCR
• The process of making many copies of a gene is called amplification of
gene.
• It is achieved through a technique called polymerase chain Reaction
(PCR).
• PCR was originally invented by kary Mullis in 1985.
• In PCR, multiple copies of the gene of interest (DNA of interest or
desirable gene) is synthesized in vitro using 2 sets of primers and the
enzyme DNA polymerase.
• Primers are small chemically synthesized oligonucleotides that are
complementary to the regions of DNA.
• PCR has 3 basic steps- Denaturation, Primer annealing and primer
extension.
(a) Denaturation
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• The target DNA is heated to high temperature (about 94 C).
• It results in separation of 2 strands.
• Each single strand of the target DNA acts as a template for DNA
synthesis.
(b) Primer annealing
• Here, 2 small chemically synthesized oligonucleotide primers (that are
complementary to the regions of DNA ) anneal (hybridize) to each of the
single –stranded template DNA at its 3’ end.
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• Annealing occurs at low temperature (40 C -60 C).
(c) Primer Extension
• During this step, deoxynucleoside triphosphate (dNTPs), Mg2+ , and
DNA polymerase are added.
• Commonly used DNA polymerase for PCR reactions is Taq Polymerase
(thermostable DNA polymerase).
• This enzyme is isolated from a bacterium ,Thermus aquaticus .
• Taq polymerase remains active during high temperature at 720C.
Other DNA polymerases used in PCR
[Link] polymerase isolated from pyrococcus furiosus.
[Link] polymerase isolated from Therococcus litoralis.
 Taq polymerase synthesizes the DNA strands complementary to
template strand. This process is called primer extension or polymerization.
• If the process of replication of DNA is repeated many times, the segment
of DNA can be amplified to approximately billion times (i.e., 1 billion
copies are made).
0
• For starting the next cycle, DNA is again heated to 94 C for denaturation
of newly synthesized DNAs.
• This is followed by annealing and extension.

• During PCR , the polymeration product (extension product) of one cycle

serves as a template for subsequent cycles and each time the amount of
DNA doubles.
• It is possible to amplify DNA segment to approximately 1 billion times
or obtain one billion copies of a DNA segment within a span of one day.
• Amplified DNA segment can now be used to ligate with the vector for
formation of rDNA and its further cloning.
• [Link] of recombinant DNA in the host cell or host Organism
• The ligated DNA is introduced in to the recipient cells by making them
“competent” to receive (take up) DNA present in its surrounding.
• The Plasmids having recombinant DNA (rDNA) and ampicillin –resistant
gene (ampR) are introduced in their medium.
• The plasmids enter the [Link] cell.
• As a result, the host cells become transformed in to ampicillin –resistant
cell.
• If the transformed cells are spread on agar plates containing ampicillin,
only transformants will grow and untransformed recipient cell will die.
• Therefore, due to ampicillin-resistant gene, one is able to select a
transformed cell in the presence of Ampicillin.
• In this case, the ampicillin resistant –gene (ampR) is called as “selectable
marker”.
• The transformed cells are now selected in the presence of ampicillin for
use in Bioreactor.
5. Obtaining the foreign gene product
• When a recombinant DNA (rDNA) is transferred in to a host cell
(bacterial or plant or animal cell), the alien DNA is multiplied.
• The foreign gene (alien DNA) gets expressed under appropriate
conditions.
• It is important to know the technological details and optimum conditions
under which a foreign gene is expressed in host cells.
• The ultimate aim of recombinant biotechnology is to produce a desired
protein useful to humans.
• The protein produced by the expression of a protein –encoding gene
through rDNA technology inside heterologous host, is called recombinant
protein.
• The cells containing cloned gene of interest may be grown on a small
scale in the laboratory.
• The cultures may be used for extracting and purifying the desired protein.
• The cells can also be multiplied in a continuous process, where the used
medium is taken out from one side and fresh medium is added from the
other side to maintain the cells in their physiologically most active
exponential phase (phase of rapid multiplication of cells).
• Through this type of culturing method, large quantity of biomass is
produced which gives rise to higher yields of desired protein.
• Small volume cultures cannot yield the products in large quantities.
Therefore, bioreactors are used to obtain the products in large
quantities.
Bioreactor
• The device in which the biochemical process is carried out by using
living cells and their growth medium, is called bioreactor or fermenter.
 • Bioreactor is used to obtain the foreign gene products in large quantities.
• Large volumes (100-1000 litres) of culture can be processed in a
bioreactor.
Types of Bioreactors
Bioreactors are of different types. The important types of bioreactors are
the following.
1. Stirring type Bioreactor.
2. Pressure cycle Bioreactor.

• The most commonly used bioreactor is stirring type [Link] may be

of different types.
• Stirred –Tank bioreactor is the common stirring type bioreactor.

Stirred –Tank Bioreactor

• Stirred –tank bioreactor used in biotechnology is generally 100-1000 litre
cylindrical vessel (container) with a curved base to facilitate mixing of
reactor contents.
• The culture medium containing all the nutrients, salts, vitamins,
hormones, etc., is added along with inoculum of transformed cells with
recombinant DNA.
• It has a stirrer which facilitates even mixing and oxygen availability
throughout the bioreactor. This bioreactor has a number of systems.
• Agitator System
• Oxygen delivery system
• Foam control system
• Temperature control system
• pH control system
• Sampling ports
Small volumes of culture can be taken out periodically
through sampling ports.
Modern stirred –tank bioreactor has adequate arrangement for
aeration, temperature, and pH control .For proper aeration ,air can be
forced in at the bottom of the tank through a porous ring, called sparger ,
by the process called sparging.
• There is an outlet to remove air and waste gases at the top of the tank.
• The top of the tank also has a number of inlet tubes called ports.
• Materials can be introduced or withdrawn through the ports.

Significances of stirred tank bioreactor

1. Stirred –tank bioreactor is a well –tired and tested design for large-scale
production of micro-organisms under aseptic and controlled environment
for a number of days.
2. It is provided with many controls for the monitoring of physical,
chemical, and biological parameters that affect the growth of cells.

6 .Downstream processing
• The process of separation and purification of a biosynthetic product is
called downstream processing.
• After the formation of a biosynthetic product, it is subjected to a series of
processes before it is ready for marketing as a finished product.
• The important steps in downstream processing are the following.
1. Separation of gene product.
2. Purification of gene product.
3. Addition of suitable preservatives.
4. Clinical trials for medicinal purposes (drugs).
5. Quality control test (it varies From product to product).