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 1 Biotechnology Principles
and Processes
SCIENTISTS WINDOW
Scientists Contribution
Karl Ereky Coined term biotechnology
Arber, Nathan, Smith Discovered the restriction endonucleases (molecular
scissors) that can cleave the DNA at specific sites (Nobel
Prize-1978).
▪▪ Arber ▪▪ He told that these enzymes use to bind at specific
points on DNA and are responsible for its cleavage and
modification (methylation).
▪▪ Nathan ▪▪ He used the restriction enzymes in some of his pioneer
works in the field of genetics
▪▪ Smith ▪▪ He told about the discovery of first restriction
endonuclease- Hind II (from Haemophilus influenzae)
and mechanism of its action
Paul Berg He has introduced a gene of SV-40 into a bacterium with
the help of lambda phage; called as the father of genetic
engineering (Nobel Prize-1980)
Kornberg Discovered DNA polymerase-I from E.coli and
synthesized first synthetic DNA from a mixture of
deoxyribonucleotides, DNA polymerase enzyme, metal
ions and a segment of viral DNA
Kary Mullis Discovered PCR
Khorana Synthesized the first artificial gene without using a
template
Ochoa Synthesized the first artificial RNA
Barbara Mc Clintock Discovered transposons or jumping genes in Maize
Beadle and Tatum Proposed one gene one enzyme hypothesis

♦♦ Biotechnology, the 20th century off-shoot of modern biology, changed our daily life
as its products brought qualitative improvement in health and food production.
♦♦ Herbert Boyer: He performed studies on a couple of restriction enzymes of the
E. coli bacterium with especially useful properties. Boyer observed that these
enzymes have the capability of cutting DNA strands in a particular fashion, which
left what has became known as ‘sticky ends’ on the strands. These clipped ends
made pasting together pieces of DNA a precise exercise.
♦♦ Stanley Cohen: He was studying small ringlets of DNA called plasmids (extra
chromosomal, circular, double stranded DNA also called as gene taxi) which float
about freely in the cytoplasm of certain bacterial cells and replicate independently
from the coding strand of DNA. Cohen had developed a method of removing these

1
 plasmids from the cell and then reinserting them in other cells.
♦♦ Old definition of Biotechnology: It deals with techniques of using live organisms
(making curd, antibiotics, bread or wine with the help of microbes) or enzymes
from organisms to produce products and processes useful to humans.
♦♦ Modern view of biotechnology: The production technologies based on genetic
engineering is the modern view such as the production of human insulin from
transgenic E.coli.
♦♦ In vitro fertilization leading to a ‘test-tube’ baby, synthesizing a gene and
using it, developing a DNA vaccine or correcting a defective gene, are all part of
biotechnology.
♦♦ Biotechnology provides the base for genetic engineering or Recombinant DNA
technology (RDT) which can manipulate an organism’s genetic material to make
many drugs, enzymes, disease resistant plants etc.
♦♦ Standard definition of Biotechnology given by EFB (European Federation of
Biotechnology): ‘The integration of natural science and organisms, cells, parts
thereof, and molecular analogues for products and services’. This definition
includes both traditional as well as modern molecular view.
PRINCIPLES OF BIOTECHNOLOGY

♦♦ Among many, the two core techniques that enabled birth of modern biotechnology
are:
(i) Genetic engineering: Techniques to alter the chemistry of genetic material
(DNA and RNA), to introduce these into host organisms and thus change the
phenotype of the host organism.
For example- If you want to make human insulin by using a simple bacterium
then what you have to do is to clone a piece of human insulin gene (gene of
interest) from human genome and insert that gene into the genome of bacteria.
When the bacteria will replicate (divide/to make copies), our gene of interest
will also transcribe (formation of mRNA from DNA) and translate (formation
of protein from mRNA) to form a functional protein which is going to be our
human insulin.
(ii) Bioprocess Engineering: Maintenance of sterile (microbial contamination-
free) ambience in chemical engineering processes to enable growth of only
the desired microbe/eukaryotic cell in large quantities for the manufacture of
biotechnological products like antibiotics, vaccines, enzymes, etc. This aspect
is related to the pure culturing. If we want to do good piece of research work
then it becomes very important to keep the system free from contamination
(from other microbes and pathogens). If we are working on E.coli then we want
its pure culture (all bacterium are of same type and are generated from single
bacterium) to get desired outcomes.
♦♦ There are several advantages of sexual reproduction over asexual reproduction.

2
 Sexual reproduction provides opportunities for variations and formulation of
unique combinations of genetic setup, some of which may be beneficial to the
organism as well as the population. Asexual reproduction preserves the genetic
information, while sexual reproduction permits variation.
♦♦ Traditional hybridisation procedures used in plant and animal breeding, very often
lead to inclusion and multiplication of undesirable genes along with the desired
genes (due to lack of selectivity of specific characters). The techniques of genetic
engineering which include creation of recombinant DNA (formed by inserting gene
of interest into the genome of host- since it requires combining of genome of 2
organisms, so it is called a recombinant DNA), use of gene cloning and gene
transfer, overcome this limitation and allows us to isolate and introduce only one or
a set of desirable genes (gene of interest which are chosen by us which makes this
process to yield desirable products only) without introducing undesirable genes
into the target organism.
What can be the fate of a piece of DNA which is transferred into an alien organism?
♦♦ Most likely, this piece of DNA would not be able to multiply itself in the progeny
cells of the organism.
♦♦ When the piece of DNA gets integrated (incorporated in the DNA of host) into the
genome of the recipient, it may multiply and be inherited along with the host DNA.
This is because the alien piece of DNA has become part of a chromosome, which
has the ability to replicate. In a chromosome there is a specific DNA sequence
called the origin of replication/ori, which is responsible for initiating replication.
Therefore, for the multiplication of any alien piece of DNA in an organism it needs
to be a part of a chromosome(s) which has a specific sequence known as ‘origin
of replication’. Thus, an alien DNA is linked with the origin of replication, so that,
this alien piece of DNA can replicate and multiply itself in the host organism. This
can also be called as cloning or making multiple identical copies of any template
DNA.
PROCESS OF GENE CLONING:

1. Gene of interest is inserted into vector (usually a plasmid) to make rDNA

(recombinant DNA) molecule.
2. The vector containing the gene of interest acts as a vehicle which transports our
gene of interest into host cell (usually a bacterium).
3. Vector replicates its genome in the host cell and thus the gene of interest is also
replicated with the vector genome.
4. rDNA got distributed to the daughter cells when the host cell divides and further
replication of vector occurs which make multiple copies of gene of interest too.
5. After thousands of divisions, a colony (clone) of identical host cells is formed
which are carrying one or more copies of our rDNA molecule and thus our gene of
interest.

3
 Fig: Process of gene cloning explained
CONSTRUCTION OF FIRST RECOMBINANT DNA (rDNA) MOLECULE:
♦♦ The construction of the first recombinant DNA emerged from the possibility of
linking a gene encoding antibiotic resistance with a native plasmid (autonomously
replicating circular extra-chromosomal DNA) of Salmonella typhimurium
(causative agent of Typhoid) to make this bacterium resistant of antibiotics.
♦♦ Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the
antibiotic resistance gene by cutting out a piece of DNA from a plasmid which
was responsible for conferring antibiotic resistance which can be inserted in other
bacterium to provide resistance against antibiotics.
♦♦ The cutting of DNA at specific locations by recognizing specific sequence of
nitrogenous bases became possible with the discovery of the so-called ‘molecular
scissors’ or restriction enzymes.
♦♦ The cut piece of DNA (conferring antibiotic resistance) was then linked with the
plasmid DNA. These plasmid DNA act as vectors/vehicles to transfer the piece of
DNA attached to it which is basically our gene of interest into the host organism
just like the mosquito acts as an insect vector to transfer the malarial parasite into
human body.
♦♦ The linking of antibiotic resistance gene with the plasmid vector became possible
with the enzyme DNA ligase, which acts on cut DNA molecules and joins their
ends. This makes a new combination of circular autonomously replicating DNA
created in vitro and is known as recombinant DNA (it is called as recombinant

4
 DNA as it is formed up by the combination of plasmid genome and our gene of
interest).
♦♦ When this DNA is transferred into Escherichia coli, a bacterium closely related to
Salmonella, it could replicate using the new host’s DNA polymerase enzyme and
make multiple copies. The ability to multiply copies of antibiotic resistance gene in
E. coli was called cloning of antibiotic resistance gene in E. coli.
♦♦ There are three basic steps in genetically modifying an organism-
(i) Identification of DNA with desirable genes (gene of interest)
(ii) Introduction of the identified DNA into the host
(iii) Maintenance of introduced DNA in the host and transfer of the DNA to its
progeny.
TOOLS OF RECOMBINANT DNA TECHNOLOGY
♦♦ Key tools in genetic engineering or recombinant DNA technology are restriction
enzymes (for precise cutting at specific points), polymerase enzymes (for
replication), ligases (for joining of DNA molecules to create recombinant DNA),
vectors (for transferring the gene of interest into host organism) and the host
organism (to clone the gene of interest).
♦♦ Let’s discuss about each one of them-
1. Restriction enzymes: In the year 1963, the two enzymes responsible for restricting
the growth of bacteriophage (viruses that infects bacteria) in Escherichia coli
were isolated. One of these added methyl groups (methylation) to DNA, while
the other cut DNA (cleavage). The later was called restriction endonuclease.
They are basically derived from prokaryotes/bacteria as they provide a defense
mechanism against bacteriophages. These enzymes use to cleave the DNA of
bacteriophages and restrict their growth.
• The first restriction endonuclease was Hind II, whose functioning depended
on a specific DNA nucleotide sequence was isolated and characterized five
years later. It was found that Hind II always cut DNA molecules at a particular
point by recognizing a specific sequence of six base pairs. This specific base
sequence is known as the recognition sequence for Hind II.
• Besides Hind II, today we know more than 900 restriction enzymes that
have been isolated from over 230 strains of bacteria each of which recognise
different recognition sequences.
NAMING OF RESTRICTION ENZYMES (e.g. EcoRI comes from E.coli RY 13)
Character in Name Derived from Comments
E Escherichia Name of Genus (first letter)
Co coli Name of Species (next two letters)
RY RY named strain Strain of bacterium (R stands for rough which means
absence of polysaccharide coat on surface which makes
it non virulent)
13 Order number (in Order in which enzyme is isolated from a particular
Roman e.g. I in strain of bacterium
case of EcoRI)

5
 • Restriction enzymes belong to a larger class of enzymes called nucleases.
These are of two kinds namely exonucleases and endonucleases. As the name
indicated, endo means ‘in between’ so it will make cuts at specific positions
within (in between) the DNA and exonucleases will remove nucleotides from
the ends of the DNA.

Fig: It is clearly depicted that the restriction endonucleases EcoRI use to cleave
the DNA strands at specific recognition sequence (palindromic sequence) which is
5’-GAATTC-3’

Fig: Action of restriction enzyme

Conceptual Clarity
One thing which is noteworthy in the study of restriction endonucleases is that they
always use to cleave the DNA strands at very same recognition sequence. It means
that the sticky ends formed by the restriction endonuclease are also going to be same
in all the DNA strands cleaved by it. So it becomes very necessary that our gene of
interest and the vector must be cleaved with the same restriction endonucleases so

6
that same kind of sticky ends can be formed and thus the complimentary sequences
can easily pair up with each other to form the recombinant DNA.
As shown in the figure above EcoRI is cleaving both vector and foreign DNA to
generate sticky ends which can pair up by complimentary base pairing (Adenine pairs
with Thymine with the help of 2 hydrogen bonds and the Cytosine use to pair up with
Guanine with the help of 3 hydrogen bonds) to form the recombinant DNA.

Fig: sticky end formation by restriction endonuclease. Each restriction endonuclease

functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific
recognition sequence (can be 4-8 nucleotide long), it will bind to the DNA and cut
each of the two strands of the double helix at specific points in their sugar -phosphate
backbones. Each restriction endonuclease recognizes a specific palindromic (a
sequence which comes out to be exactly same when read from any of the side, such as
in MALAYALAM word- the sequence of alphabets remains same when read in any of
the direction) nucleotide sequences in the DNA.
• Since all the restriction enzymes are not from the same strain of bacterium so
different restriction enzymes will obviously have their specific recognition site
at which it will cleave the DNA strand.
• The table given below will depict various strains and restriction enzymes isolated
from them. All of them are having their specific recognition sequences and just
by looking at the cleavage position, we can make some major conclusions as
well.
Recognition sequence and site
Restriction
Source of cleavage which is marked
enzyme
by (|) the sign inside bracket
5’-A-G-|-C-T -3’
Alu I Arthrobactor luteus
3’- T-C -|-G-A-5’
5’-G-|-G-A-T-C-C -3’
Bam H I Bacillus amyloliquefaciens H
3’- C-C-T-A-G-|-G-5’
5’-G-|-A-A-T-T-C -3’
Eco R I Escherichia coli RY 13
3’- C-T-T-A-A-|-G-5’
5’-|-C-C-T-G-G-3’
Eco R II Escherichia coli R 245
3’- G-G-A-C-C-|-5’

7
 Recognition sequence and site
Restriction
Source of cleavage which is marked
enzyme
by (|) the sign inside bracket
5’-G-G-|-C-C -3’
Hae III Haemophilus aegyptius
3’-C- C -|-G-G -5’
5’-A-|-A-G-C-T-T -3’
Hin d III Haemophilus influenzae Rd
3’- T-T-C-G-A-|-A-5’
5’-G-T-C -|-G-A-C -3’
Hin d II Haemophilus influenzae Rd
3’-C-A-G-|-C-T-G-5’
5’-G-|-T-C-G-A-C -3’
Sal I Streptomyces albus
3’- C-A-G-C-T-|-G-5’
5’-A-G-T-|-A-C-T-3’
Sca I Streptomyces caespitosus
3’- T-C-A-|-T-G-A-5’
5’-C- C –C-|-G-G-G -3’
Sma I Serratia marcescens
3’-G-G-G -|-C-C-C-5’

Conceptual Clarity
▪▪ As I’ve told, let’s make some conclusions from the table shown above to learn
some new concepts. Among the restriction enzymes mentioned above in the table,
some use to cut the strand of DNA a little away from the centre of the palindrome
sites, but between the same two bases on the opposite strands. This leaves single
stranded portions at the ends. There are overhanging stretches called sticky ends
on each strand. These are named so because they form hydrogen bonds with their
complementary cut counterparts. This stickiness of the ends facilitates the action of
the enzyme DNA ligase. Examples are Bam H I, Eco R I, Hin d III, Sal I in which
the enzyme is not cleaving the DNA exactly at the centre of palindromic sequence
and thus single stranded sticky ends are produced.
▪▪ In some cases the restriction enzymes use to cut the DNA in the centre of palindromic
sequences. In this case, no single strands are left and hence there will be no sticky
ends so the ends are called to be blunt or flush ends. Examples are Sma I, Sca I,
Hin d II, Hae III, Alu I.
▪▪ It is amazing to note that both Hin d II and Hin d III are produced by Haemophilus
influenzae but both produce different ends (Hin d II produce blunt ends and Hin d III
produces sticky ends).
▪▪ It is also noteworthy that some restriction enzymes have recognition sequence
of 4 nucleotides and some have 6 nucleotides long signal sequence. In all those
cases, there is one exception which is not necessarily palindromic as it is having 5
nucleotide long recognition sequence (Eco R II).
• Restriction endonucleases are used in genetic engineering to form ‘recombinant’
molecules of DNA, which are composed of DNA from different sources/
genomes.
• When cut by the same restriction enzyme, the resultant DNA fragments have
the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end)
using DNA ligases.
• If the gene of interest and the vector are cleaved by different restriction
enzymes then they cannot be joined to make rDNA as the ends of two DNA
segments are not going to be same.

8
 2. Ligase:
• Ligase enzyme basically joins the two DNA molecules from different sources
(gene of interest and vector DNA) but cleaved by same restriction enzyme.
• It usually makes the phosphodiester bond between the adjacent nucleotides
which is an energy requiring process.
• Enzyme generally used in genetic engineering is T4 ligase which is encoded
by the genome of T4 phage (even phage virus called Enterobacteria phage T4
which infects E.coli).
• Ligases are also called as genetic gum or glue.
Out of The Box
Alkaline Phosphatase enzymes are helpful in removing the phosphate group from
the 5’ end of DNA molecule, leaving a free 5’ hydroxyl group. It can be isolated from
calf’s intestine or from bacteria. It is used to prevent unwanted self ligation of vector
DNA molecules in process of genetic engineering.

Fig: Action of ligase enzyme and overview of genetic engineering

3. DNA polymerase: It is employed to polymerize replication of DNA on DNA

template or complementary DNA (cDNA) and that’s why it is named as DNA
dependent DNA polymerase.
• It uses to synthesize DNA in 5’→ 3’ direction. It can also show proof reading
(detection of errors) activity from 3’→5’ direction.
• DNA polymerase firstly investigated by Kornberg in E.coli and is now known
as DNA polymerase-I which is usually employed in genetic engineering.

9
4. Cloning Vectors: Cloning vectors are basically used to transfer the gene of
interest and replicate into the host cell.
• Plasmids and bacteriophages have the ability to replicate within bacterial
cells independent of the control of chromosomal DNA.
• Bacteriophages because of their high number per cell have very high copy
numbers of their genome within the bacterial cells. Some plasmids may have
only one or two copies per cell whereas others may have 15-100 copies per
cell. Their numbers can go even higher. If we are able to link an alien piece of
DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal
to the copy number of the plasmid or bacteriophage.
• Plasmids (they are most commonly used in rDNA technology and they are
extra chromosomal, self replicating, circular, double stranded DNA molecules),
bacteriophage, cosmids, phagemids, Yeast artificial chromosomes (YACs),
Bacterial artificial chromosomes (BACs), transposons (transposable genetic
elements), shuttle vectors etc. are used as vectors in genetic engineering.
• Vectors used at present, are engineered in such a way that they help in:
- Easy linking of foreign DNA
- Selection of recombinants from non-recombinants.
• The following are the features that are required to facilitate cloning into a vector
are-
(i) Origin of replication (ori): replication starts here and any piece of DNA
when linked to this sequence can be made to replicate within the host cells.
This sequence is also responsible for controlling the copy number of the
linked DNA. So, if one wants to recover many copies of the target DNA it
should be cloned in a vector whose origin support high copy number.
(ii) Selectable marker: selectable markers help in identifying and
eliminating nontransformants and selectively permitting the growth of
the transformants.
Transformation is a procedure through which a piece of DNA is introduced
in a host bacterium.
Normally, the genes encoding resistance to antibiotics such as ampicillin,
chloramphenicol, tetracycline or kanamycin etc., are considered useful
selectable markers for E. coli. The normal E. coli cells do not carry resistance
against any of these antibiotics.

10
 Colour Reaction To Select Transformants
Due to inactivation of antibiotics by antibiotic resistance gene, the selection of
recombinants becomes quite hectic process because it requires simultaneous plating on
two plates having different antibiotics. Thus alternative selectable marker is developed
to differentiate recombinants and non-recombinants on the basis of their ability to
produce colour in the presence of a chromogenic (colour producing) substance such
as β-galactosidase.
When a recombinant DNA is inserted in the coding sequence of an enzyme
β-galactosidase, the gene (lac Z) responsible for the synthesis of β-galactosidase is
inactivated. Since this inactivation occurred due to the insertion of rDNA in the coding
region of β-galactosidase so it is called insertional inactivation. If the plasmid in the
bacterium does not have an insert, the presence of activated β-galactosidase gene will
give blue coloured colonies. Presence of insert results into insertional inactivation
of the β-galactosidase and, therefore, the colonies do not produce any colour, these
colonies are marked as recombinant colonies in which the rDNA has done inactivation.
Thus the selection becomes a single step process in case of pUC8 (a plasmid) which
use this mechanism of recombinant selection.
(iii) Cloning sites: In order to link the alien DNA, the vector needs to have very
few, preferably single, recognition sites for the commonly used restriction
enzymes.
Presence of more than one recognition sites within the vector will generate
several fragments, which will complicate the process of gene cloning.
Selection of transformants by repetitive/simultaneous plating method:
The ligation of alien DNA is carried out at a restriction site present in one of
the two antibiotic resistance genes in pBR322. For example, the ligation of a
foreign DNA at the Bam H I site of tetracycline resistance gene in the vector
pBR322 (bears genes for resistance against two antibiotics i.e. ampicillin and
tetracycline). The recombinant plasmids will lose tetracycline resistance
due to insertion of foreign DNA but can still be selected out from non-
recombinant ones by plating the transformants on ampicillin containing
medium. The transformants growing on ampicillin containing medium are
then transferred on a medium containing tetracycline. The recombinants will
grow in ampicillin containing medium but not on that containing tetracycline
(since this resistance is now inactivated by the insertion of rDNA so the
resistance to ampicillin is left only). But, non recombinants will grow on
the medium containing both the antibiotics (both the resistance genes are
active so that the bacteria can survive on both antibiotics). In this case, one
antibiotic resistance gene helps in selecting the transformants (ampicillin),
whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’
(tetracycline)of alien DNA, and helps in selection of recombinants.
TYPES OF VECTORS USED IN GENETIC ENGINEERING:
(i) Plasmid: It is extra chromosomal, independently replicating, circular, double
stranded DNA molecule which is found in many bacteria and some yeast. They
may occur as one or two copies or in multiple copies (500–700 in case of pUC8
plasmid) inside the host organism.

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 Plasmid DNA lack introns and is naked (without histones).

Fig: pBR322- an ideal plasmid vector

NOMENCLATURE of pBR322
Element of Name What it denotes
p Plasmid
BR Bolivar and Rodriguez who constructed this plasmid
Number given to distinguish this plasmid from other
322
developed in the same laboratory
• E. coli cloning vector pBR322 shows restriction sites (Hind III, EcoR I, BamH
I, Sal I, Pvu II, Pst I, Cla I), ori and antibiotic resistance genes (ampR and tetR)
in the diagram. rop codes for the proteins involved in the replication of the
plasmid.
• Features of pBR322: it has following regions-
- Origin of replication (Ori ): replication initiation site
- Antibiotic resistance genes: 2 antibiotic resistance genes are present which
are ampR and tetR for ampicillin resistance and tetracycline resistance
respectively. Due to the presence of 2 antibiotic resistance genes, the
selection of recombinants is a two step procedure where simultaneous
plating needs to be done which is a demerit of pBR322.
- Unique recognition sites for different restriction endonucleases: Pst I and
Pvu I are located within the ampR gene and Bam H I, Sal I, etc. are within
tetR gene. Some other restriction sites are Eco R I, Cla I, Hind III, Pvu II.
- Its size is around 4.3 Kb.
(ii) Bacteriophage: These are the viruses that infect bacteria. Bacteriophages have
quite high copy number as compared to plasmids.
• The DNA of phage undergoes replication by taking over bacterial cell
machinery and forms number of phages which burst out of the cell and
reinfect neighbouring cells to produce even more copies of rDNA.
• Lambda (λ) phage and M13 phage are most commonly used cloning vectors.
(iii) Bacterial Artificial chromosome (BAC): They can accommodate upto 350Kb
of foreign DNA. The basis of these vectors is natural or extrachromosomal
plasmid of E.coli which is called the fertility factor (F-factor).

12
 • E.coli also carries genes for the maintenance and replication of F-factor.
(iv) Yeast artificial Chromosome (YAC): They are employed to clone DNA
fragments of more than 1 Mb in size.
• YAC contains independently replicating sequence from yeast chromosome
alongwith centromere and telomere sequences.
(v) Cosmid: as the name indicates, it is formed by combining the features of ‘cos’
site of lambda phage and that of plasmid.
• It is used to clone DNA fragments upto 40-45Kb in length.
(vi) Phagemid: It is made by the combining bacteriophage and plasmid DNA as
the name indicates.
(vii) Shuttle vectors: Several vectors are constructed for the use in both eukaryotes
and prokaryotes such as E.coli. They have ori and selectable marker genes for
both prokaryotes and eukaryotes. E.g. YEp or Yeast Episomal plasmid.
(viii) Transposons: They are basically called as the transposable genetic elements
or jumping genes as they can change their position from one DNA molecule
to the other.
• Jumping genes are discovered by Barbara McClintock.
(ix) Vectors for cloning genes in plants: Agrobacterium tumifaciens, a pathogen
of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’
to transform normal plant cells into a tumor and direct these tumor cells to
produce the chemicals required by the pathogen.
• It generally enters from the wounds and transforms these cells into tumor
which is basically called as crown gall.
• The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now
been modified into a cloning vector which is no more pathogenic to the plants
but is still able to use the mechanisms to deliver genes of our interest into a
variety of plants.

Fig: Formation of crown gall and tumor development

(x) Vectors for cloning genes in animals: Retroviruses in animals have the
ability to transform normal cells into cancerous cells.

13
 • Retroviruses have also been disarmed and are now used to deliver desirable
genes into animal cells. So, once a gene or a DNA fragment has been ligated
into a suitable vector it is transferred into a bacterial, plant or animal host
(where it multiplies).
5. Competent Host (For Transformation with Recombinant DNA): Since DNA
is a hydrophilic molecule, it cannot pass through cell membranes (lipid bilayer
is basically rich in hydrocarbons which are non polar and are thus hydrophobic).
• In order to force bacteria to take up the plasmid, the bacterial cells must first be
made ‘competent’ to take up DNA. This is done by treating them with a specific
concentration of a divalent cation, such as calcium as in CaCl2 (50mM for 12-
24 hour which will precipitate the DNA outside the cell), which increases the
efficiency with which DNA enters the bacterium through pores in its cell wall.
• Recombinant DNA can then be forced into such cells by incubating the cells
with recombinant DNA on ice, followed by placing them briefly at 42°C
(heat shock) for 2 minutes, and then putting them back on ice. This enables
the bacteria to take up the recombinant DNA. This process is termed as
transformation.
• Other modes of injecting DNA into host cell:
(i) Micro-injection: In this process, the recombinant DNA is directly injected
into the nucleus of an animal cell.
• It is used to transfer the gene directly in oocytes, eggs and embryo.

Fig: Method of micro-injection; suction is present in the micropipette/holding pipette

shown in image help us to hold the host cell correctly and thus increases the ease of
transferring the rDNA into host cell.
(ii) Gene gun or Biolistic: In this process, DNA coated onto microscopic pellets
of gold or tungsten is literally shot with high velocity into target cells.

14
 Fig: Structure of a gene gun (this method is quite suitable for plants)
(iii) Disarmed pathogen: they are allowed to infect the cell, transfer the
recombinant DNA into the host. For example the tumor inducing (Ti) plasmid
of Agrobacterium tumifaciens.
(iv) Direct DNA Injection in skeletal muscles: it led to the possibility of using
Gene as vaccines or genetic immunization.
(v) Electroporation: as the name indicates, elecro means electrical impulses and
poration means pore formation. So in this method short electrical impulses
induce transient pores in the host cell membrane through which the DNA
molecules are passed into the host cells.
(vi) PEG: Polyethylene glycol helps the DNA to enter into the host cell by
promoting protoplast fusion.
(vii) Liposome mediated gene transfer: DNA is enclosed within lipid bag.
Liposome is formed when the lipid bilayer folds upon itself. The cavity
surrounded by lipid bilayer can be used to transfer DNA or drug (drug delivery
as liposome can be easily transferred to cells and does not cause side effects as
it is a biological polymer).
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
♦♦ Recombinant DNA technology involves several steps in specific sequence such as
isolation of DNA, fragmentation of DNA by restriction endonucleases, isolation of
a desired DNA fragment, ligation of the DNA fragment into a vector, transferring
the recombinant DNA into the host, culturing the host cells in a medium at large
scale and extraction of the desired product.
♦♦ Let us examine each of these steps in detail.
1. Isolation of the Genetic Material (DNA): Nucleic acid is the genetic material of
all organisms without exception. In majority of organisms this is deoxyribonucleic
acid or DNA (except some viruses where RNA is the genetic material).
• In order to cut the DNA with restriction enzymes, it needs to be in pure form,
free from other macro-molecules.
• The cells are first homogenized in centrifuge at high speeds. Differential
centrifugation removes all the heavier parts in each set of rotation (heavier
particles will be more attracted by the gravitational force and are present at

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 base and lighter one will obviously float as supernatant, thus separated in each
round).
• Since the DNA is enclosed within the membranes, we have to break the cell
open to release DNA along with other macromolecules such as RNA, proteins,
polysaccharides and also lipids. This can be achieved by treating the bacterial
cells/plant or animal tissue with enzymes such as lysozyme (for bacteria),
cellulase (for plant cells), chitinase (for fungus).
• The RNA can be removed by treatment with ribonuclease whereas proteins
(histones and non histone proteins which help in the DNA packaging) can be
removed by treatment with protease.
• Carbohydrates can be digested by amylase or polysaccharidase.
• Other molecules can be removed by appropriate treatments and purified DNA
ultimately precipitates out after the addition of chilled ethanol.
TYPE OF CELL WALL AND DIGESTIVE ENZYME USED
Type of Digestive
Cell wall type Monomer of cell wall
cell enzyme used
Plant cell Cellulase Cellulose β-D-Glucose
Fungi Chitinase Chitin N-acetyl Glucosamine
Bacteria Lysozyme Peptidoglycan NAG+NAM which are N-acetyl
Glucosamine and N-acetyl
Muramic acid

Fig: Separated DNA can be seen as collection of fine threads in the suspension. The
DNA that separates out can be removed by spooling as shown here.
2. Cutting of DNA at Specific Locations: Restriction enzyme digestions are
performed by incubating purified DNA molecules with the restriction enzyme, at
the optimal conditions for that specific enzyme.
• Agarose gel electrophoresis is employed to check the progression of a restriction
enzyme digestion.
• DNA is a negatively charged molecule (due to phosphate group), hence it
moves towards the positive electrode (anode). The process is repeated with the
vector DNA also.
• The vector and the gene of interest must be cleaved with same restriction
enzyme as discussed earlier.

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 3. Isolation and separation of DNA fragments: The cutting of DNA by restriction
endonucleases results in the fragments/pieces of DNA. These fragments can be
separated by a technique known as gel electrophoresis.
• The negatively charged DNA will move toward anode under an electric field
through a medium/matrix (usually agarose which is a natural polymer extracted
from sea weeds).
• The DNA fragments separate (resolve) according to their size through sieving
effect provided by the agarose gel. Hence, the smaller the fragment size, the
farther it moves from the well.

How Gel Separates DNA Fragments

Agarose is basically a linear polymer of Agarobiose (D-Galactose and 3,6 anhydro-L-
Galactose) which is extracted from sea weeds. This polymer basically forms the pores
through which DNA will pass. Now it is quite obvious that if more concentration
of agarose gel is taken then it will be having more cross links. This extensive cross
linking will obviously decrease the pore size. So we can conclude that concentration
of agarose gel is inversely proportional to pore size. The pore will not let bigger DNA
molecules to pass through it and thus large molecules cannot move much far from the
well but the smaller fragments can run faster as they can easily pass though pores.
That’s how the DNA fragments of different sizes are separated by gel electrophoresis.

• The separated DNA fragments can be visualized only after staining the DNA
with a compound known as ethidium bromide (DNA binding dye) followed
by exposure to UV radiation (by UV transluminator) as we cannot see pure
DNA fragments in the visible light and without staining.
• We can see bright orange coloured bands of DNA in an ethidium bromide
stained gel exposed to UV light.

Fig: Agarose gel electrophoresis

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 Conceptual Clarity
Let’s make some important conclusions from the diagram of gel electrophoresis-
▪▪ Lane/Well no.1 has undigested DNA since it is not broken into fragments and thus
the gel electrophoresis is used to check the working of restriction endonuclease
whether it has cleaved the DNA or not.
▪▪ Lane no.2, 3 and 4 have digested DNA since they are in the form of fragments and
thus are separated on the gel.
▪▪ Smallest DNA fragments are farthest from the wells as they can pass even through
the small sized pores and thus their way is not restricted.
▪▪ Largest DNA fragments are present near the wells as they are not able to pass
through the small sized pores and they clog in the pores.

• The separated bands of DNA are cut out from the agarose gel and extracted
from the gel piece. This step is known as elution.
• The DNA fragments purified in this way are used in constructing recombinant
DNA by joining them with cloning vectors.
4. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction):
PCR was discovered by Kary Mullis in 1985. PCR and produce multiple
copies (even billions) of the gene of interest in vitro which is basically called
amplification.
• Raw materials needed in PCR-
- Two sets of primers (small chemically synthesized oligonucleotides that are
complementary to the regions of DNA). These primers provides the free 3’OH
group at which polymerization can start as polymerase can synthesize the new
DNA only in the presence of 3’OH group (polymerization cannot be started
de novo means on its own).
- Enzyme DNA polymerase- a thermostable DNA polymerase (stable at high
temperatures) is required for this process which is obviously extracted from
a bacterium living at high temperature. This polymerase was named ‘Taq’
polymerase as it was extracted from Thermus aquaticus (a thermophile)
which can survive in temperature upto 95°C.
- DNA template
- Four deoxyribonucleotides for the synthesis of new DNA.
- Ions such as Mg2+

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 Fig: Steps in PCR

♦♦ There are basically 3 steps in PCR-

(i) Denaturation: The DNA is heated at 94°C and thus it is denatured by the
breakage of hydrogen bonds between complementary base pairs. Each single
strand arised by Denaturation of DNA now acts as a template for DNA synthesis.
(ii) Annealing: The reaction mixture is now cooled at a temperature of 40-65°C
for a minute which is optimum for the binding of primer pair with the DNA
template.
(iii) Extension: it is done at 72°C where Taq polymerase synthesizes the DNA
region between the primers, using DNTPs (deoxynucleoside triphosphates) or
deoxyribonucleotides and Mg2+. It means the primers are extended towards
each other so that the DNA segment lying between the two primers is copied.
♦♦ After n number of cycles, 2n numbers of molecules are generated.
♦♦ One billion copies are made in approx 30 cycles of PCR.
Applications of PCR
▪▪ DNA Fingerprinting: PCR produces enough amount of DNA for analysis in the
DNA fingerprinting technique.
▪▪ Diagnosis of Pathogens: PCR based assays basically detects the presence of gene
sequences of the infectious agents.
▪▪ Diagnosis of Specific Mutations: It detects the specific mutation which is responsible
for causing a particular genetic disorder much before the onset of the disease.

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▪▪ Detection of Specific Microorganisms: from soil sample, water sample etc.
▪▪ Prenatal Diagnosis: As name indicates, it is used to detect genetic disease in foetus
before birth. If the disease is fatal, then abortion is suggested.
▪▪ Diagnosis of Plant Pathogens: Can detect virus and viroid based on their genetic
makeup and thus can detect the diseases caused by them.
▪▪ Palaeontology/fossil study: DNA sample from the dead fossils can be cloned via
PCR.
▪▪ Gene Therapy: monitors the gene in gene therapy experiments.
5. Ligation of DNA fragment into vector: done for further cloning purpose
6. Insertion of rDNA into host cell: Various methods such as gene gun, heat shock,
electroporation are already discussed.
• 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 then only transformants will grow and 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: rDNA should express itself to produce the
desired protein by the process of translation. Foreign gene gets expressed under
optimal conditions only which must be provided to obtain the gene product.
• After having cloned the gene of interest and having optimized the conditions
to induce the expression of the target protein, one has to consider producing it
on a large scale.
• If any protein encoding gene is expressed in a heterologous host (if we want to
synthesize an animal protein using bacteria then the bacteria will be serving as
heterologous host as the native protein is not naturally produced by that host), it is
called a recombinant protein. The cells harbouring cloned genes of interest may
be grown on a small scale in the laboratory. The cultures may be used for extracting
the desired protein and then purifying it by using different separation techniques.
• Continuous culture system: It’s an open system where the cells can be
multiplied in this system where the used medium is drained out from one
side while fresh medium is added from the other to maintain the cells in their
physiologically most active log/exponential phase. This type of culturing
method produces a larger biomass leading to higher yields of desired protein.
• Batch fermentation: Fermentation is basically done in a closed system (nothing
is changed after the initiation of process and the final product is removed after
production of ample amount of product) unlike continuous culture system
which was an open system (conditions can be altered).
• Small volume cultures cannot yield appreciable quantities of products. To
produce in large quantities, the development of bioreactors, where large
volumes (100-1000 litres) of culture can be processed, was required. Thus,

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 bioreactors can be thought of as vessels in which raw materials are biologically
converted into specific products, individual enzymes, etc., using microbial
plant, animal or human cells. A bioreactor provides the optimal conditions
for achieving the desired product by providing optimum growth conditions
(temperature, pH, substrate, salts, vitamins, oxygen, cooling agent, antifoaming
agent, aseptic conditions).

Fig: The most commonly used bioreactors are of stirring type-

(a) Simple stirred-tank bioreactor (b) Sparged stirred
• Bioreactor: A stirred-tank reactor is usually cylindrical or with a curved base to
facilitate the mixing of the reactor contents. The stirrer facilitates even mixing
and oxygen availability throughout the bioreactor. Alternatively air can be
bubbled through the reactor. Bioreactor also has an agitator system, an oxygen
delivery system, a foam control system, a temperature control system, pH
control system and sampling ports so that small volumes of the culture can be
withdrawn periodically.
Conceptual Clarity
Industrial production of products can be done in 3 stages:
▪▪ Laboratory scale process: Suitable microbe is used and optimum conditions for
growth are provided in laboratory.
▪▪ Pilot plant scale: At this stage cost and quality of the product is regularly checked
and bioreactors are used.
▪▪ Organisms can be grown here in support growth system (on solid media) or
suspended growth system (in liquid media).
▪▪ Manufacturing unit: Very large sized bioreactors are used.
8. Downstream Processing: After completion of the biosynthetic stage, the product
has to be subjected through a series of processes before it is ready for marketing
as a finished product.

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 • The processes include separation and purification, which are collectively
referred to as downstream processing.
• The product has to be formulated with suitable preservatives (to preserve them
for long time).
• Such formulation has to undergo thorough clinical trials as in case of drugs
(drugs cannot be given to humans directly so they are tested on other animals
before entering the market).
• Strict quality control testing for each product is also required. The downstream
processing and quality control testing vary from product to product.
Out of The Box
▪▪ Ri plasmid of A.rhizogenes (causing hairy root disease) also used as vector for gene
transfer to plant cell just like the Ti plasmid.
▪▪ Pflu Polymerase isolated from Pyrococus furiosus bacterium and Vent Polymerase
isolated from Thermococcus litoralis are also thermostable and can be used in PCR.
▪▪ Khorana synthesized first artificial gene without using a template. He synthesized
the gene coding for yeast alanine t-RNA, which contained only 77 base pairs which
was not functional in the living system. In 1979, Khorana synthesized a functional
tyrosine t-RNA gene of E. coli with 207 base pairs.
▪▪ RFLP/Restriction Fragment length polymorphism: They are produced by single
base alterations in the recognition sequences due to which the pattern of cut use to
be changed in DNA and fragments of variable length are produced. They are used
in DNA fingerprinting technique and they are well known markers.
▪▪ RAPD/Random Amplified Polymorphic DNA: It is PCR used in the study of
polymorphism of DNA.
▪▪ AFLP is Amplified fragment Length polymorphism.
▪▪ Agrobacterium tumifaciens is called natural genetic engineer.
▪▪ Southern blotting is used to detect DNA, Northern blotting is used to detect specific
RNA from an RNA mixture and Western blotting is used to separate proteins.

Miscellaneous topics for AIIMS, JIPMER and KVPY

♦♦ FISH: Fluorescence in situ Hybridization: This method involves the staining of
chromosome or its part by fluorescent dye which helps us to identify chromosomal
disorders and is helpful in gene mapping as well. This in situ hybridisation is
helpful in locating a particular gene on chromosome.
♦♦ Specific dyes or fluorescent molecules are added to stain specific regions by binding
onto them. Dye uses to absorb the wavelength of a particular region and emit longer
wavelength light in visible region which can be observe easily by fluorescence
microscope.
♦♦ How to create a Complementary DNA (cDNA) library: A cDNA library contains
the inserts derived from complementary DNA. cDNA library as the name indicates
will contain only complementary DNA which will be derived from the mRNA
template in a cell.
cDNA contains only coding region (introns are absent) of a genome.

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 Steps:
(i) Isolation of total mRNA of cell of interest
(ii) Reverse transcriptase is used to synthesize the complementary DNA using
mRNA template.
(iii) DNA polymerase will make dsDNA.
(iv) dsDNA will be inserted in vectors for cloning purpose.

Fig: cDNA library formation (poly-T strand is attached to poly-A segment as they
are complimentary. After this priming, the enzyme reverse transcriptase will do
its work (as the name indicates, it will convert mRNA into DNA). The resulting
structure (double stranded) is then further hydrolyzed by alkali into single strands on
which DNA polymerase will work.
• Superbug: Pseudomonas putida is genetically engineered to form the Superbug
which use to clear the oil spills that use to enter the sea ecosystem due to
which many aquatic organisms got killed. Scientists have inserted the gene for
hydrocarbon utilization in Pseudomonas so that it can utilize the hydrocarbon
petroleum (xylene, camphor, octane, naphthalene) products to clear them. A
single bug can’t feed on all these stuff so many strains are made and used to
clear the oil spills.

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