Chapter 3 Gene Cloning

Gene cloning is a customary procedure to use a gene for its

product in the biotechnology industry and various other
purposes. Traditionally, it engages the transfer of a DNA 3.1 Identification of
Candidate Gene
fragment containing the gene of interest to a host cell by a
vector so that many copies of the gene will be available for 3.2 Isolation of Nucleic
Acids
its characterisation and future application. Technological
breakthrough in the field of genetic engineering have made 3.3 Enzymes used for
Recombinant DNA
it possible to analyse DNA, isolate a specific gene from a Technology
genome, enzymatically inserting it into an autonomously
3.4 Modes of DNA
replicating vector (e.g. plasmid) to generate rDNA molecule Transfer
and ultimately introducing into host (e.g., bacteria) to
3.5 Screening and
produce a virtually unlimited number of copies (clones) of Selection
it. This chapter will expose students to all the procedures
3.6 Blotting
involved in gene cloning. Techniques
3.7 Polymerase Chain
3.1 Identification of Candidate Gene Reaction (PCR)
Over the past decades, rDNA technology has been utilised 3.8 DNA Libraries
to produce crops that are resistant to pests, diseases,
herbicides and pathogens. This is possible by manipulating
the specific gene of interest of one organism followed by
its transfer into the genome of another organism, which
upon expression results in the desired product or activity.

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 The first and most formidable problem in this process is to
identify the candidate gene in the genome of an organism.
Identification of a gene to be cloned depends upon
its significance with regard to its role in biomedical,
economical and evolutionary fields. This information
on a gene comes from its biochemical and physiological
studies. For example, the cause of diseases (diabetes
in human beings due to deficiency of insulin) or defect
in the metabolic pathways (iron deficiency leading to
chlorosis in plants) or resistance to environment (salinity
tolerance in plants) or resistance to infection (both in
plants and animals) or economically important genes
(milk protein, blood clotting factors, etc.) are candidate
genes for the improvement of human health and needs.
Once a gene of interest is identified, it is explored in
new sources and the same is cloned as mentioned in the
subsequent sections.
Searching a gene of interest is not an easy task. This
will be clear from the following example. As you know, a
haploid human genome contains approximately 3.2 billion
bp. Therefore, searching a gene of interest having a size of
3000 to 3500 bp, which is one-millionth of the genome;
is perhaps more difficult than looking for a needle in
the haystack.
There are a few methods developed to achieve this task.
One of these methods is to deduce the DNA sequence of the
gene coding for a specific polypeptide chain based on its
amino acid sequence. Another way to synthesize candidate
gene is to isolate the mRNA of the desired gene from
specific tissue, then synthesizing single stranded cDNA by
using reverse transcriptase enzyme and converting that in
to double stranded cDNA as candidate gene, which can be
cloned (as discussed in the subsequent section).

3.2 Isolation of Nucleic Acids

The first and foremost requirement for any molecular
biology experiment is isolation of nucleic acids from
organisms. Extraction of nucleic acids is encountered by
two big challenges. First one is related to their availability
in cells as DNA and RNA, both of which are present in very

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 small amounts in cells in comparison to other biological
macromolecules, such as proteins, carbohydrates and
lipids. Second, the enormous length of nucleic acids,
particularly makes it susceptible to cleavage when exposed
to harsh physical stress. In addition, the chemical bonds
by which different components of nucleic acids are joined
with each other and various groups present in them make
nucleic acid vulnerable to chemical agents.
Four important steps are involved during the extraction
of nucleic acids. The first step involves the effective rupture
of cell membrane or walls to release the nucleic acids and
other cellular molecules. The second step involves the
protection of nucleic acids from their respective degrading

The cells and tissues are broken

down in a lyses buffer Chilled ethanol
Centrifuged Chilled ethanol
Chloroform Chloroform Centrifuged
Ground up cells Aqueous phase is
Phenol Isoamyl Phenol Isoamyl
transferred pipatted to another tube
alcohol alcohol

Aqueous
Nucleic acid phase
Centrifuged transferred (DNA)

Aqueous phase RNase

(Nucleic acids) added
CTAB, SDS Interface
buffer, EDTA (Lysed proteins)
Nucleic acid
Organic Phase pellet
Cell lysate (Broken down proteins, lipids, DNA
and other cell debris)

Fig. 3.1: Steps involved in the isolation of DNA

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 enzymes, which are released in the isolation medium
with other proteins. In the third step, the nucleic acids
are separated from other molecules. In the fourth and the
last step, the isolated nucleic acids are precipitated and
concentrated by adding ethanol or isopropanol.
Although, chemical and physical properties of nucleic
acids are similar in all organisms, the outer boundary of
cells differs from one organism to another. Therefore, in
order to disrupt the cell boundaries for releasing nucleic
acids into extraction medium, different strategies are
adopted. Animal cells have plasma membrane that can
be easily disrupted. On the contrary, plant cells and
bacteria are protected by tough layers (e.g., cell wall),
which need different approaches for their lysis. These
include homogenisation, grinding, sonication or enzymatic
treatment. Such mechanical or enzymatic treatment
ruptures plasma membrane or cell wall so that nucleic
acids get released from cells and exposes them to nuclease
enzymes (deoxyribonuclease and ribonuclease), which are
also released simultaneously.

Isolation of DNA
As bacterial cells have little structure beyond the cell wall
and cell membrane, isolating DNA from them is much
easier. An enzyme called lysozyme digest the peptidoglycan,
the main component of bacterial cell wall. Detergents
like sodium dodecyl sulphate (SDS) is used to lyse the
cell membranes by disrupting the lipid bilayer. Plant
and animal cells are ground to release the intracellular
components. Plant cells are mechanically ruptured in a
blender to break open the tough cell walls. For isolation
of DNA from plant cells, cetyl trimethyl ammonium
bromide (CTAB) is used as detergent (a cationic detergent).
Plant cells have high concentration of polysaccharide
and polyphenols in comparison to animal cells and pose
problems during isolation of DNA. The solubility of DNA
and polysaccharides to CTAB depends on ionic strength of
the solution. At low ionic strength, DNA is soluble in CTAB
solution while polysaccharides are insoluble; whereas
at high ionic strength, polysaccharides are soluble and
DNA is insoluble. In addition, being a detergent, it also

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 lyses cell wall. Both the molecules are separated based
on their differential affinity to CTAB. Addition of polyvinyl
pyrrolidone (PVP) to CTAB extraction medium neutralises
phenols. Soluble DNA present in supernatant is extracted
with chloroform-isoamyl solution. DNA present in aqueous
phase is precipitated using ethanol or isopropanol. In case
of animal cells, the cell membrane is disrupted by detergent
to release the intracellular components.
The cells and tissues from which nucleic acids are to be
extracted are broken down in a medium either mechanically
or enzymatically. The media is usually a buffer having
mild alkaline pH with minimum ionic strength (0.05 M)
containing chelating agent ethylene diamine tetraacetic
acid (EDTA). The mild alkaline pH facilitates the reduction
of electrostatic interaction between DNA and basic proteins
(histones) released during cell disruption. Chelating of
divalent cations particularly Mn2+ and Mg2+ prevents the
action of nucleases. Further, inhibition of their activities
is achieved due to alkaline pH of the buffer. In addition,
chelating of divalent cations prevents the formation of their
respective salts with phosphate groups of nucleic acids.
The next step is to separate nucleic acids from its
bound proteins. This is achieved by decreasing interaction
between proteins and nucleic acids so that nucleic acids
are free of proteins, by exposing to detergents, like SDS, an
anionic detergent. Exposure to SDS makes all the protein
molecules anionic. Consequently, basic proteins that
are positively charged and bound to negatively charged
nucleic acids become negatively charged and dissociate
from the nucleic acids. In addition, SDS also prevents the
activities of nucleases thereby giving additional protection
to nucleic acids from nucleases. Then sodium chloride is
added to the medium at high concentration. Increased salt
concentration diminishes the ionic interaction between
DNA and cations thus ensuring complete dissociation
of DNA and protein complexes. Deproteinisation of the
medium is achieved by exposing it to chloroform and
isoamyl alcohol. These solvents are non-polar in nature
when it is added to the medium that is polar in nature and
subjected to centrifugation, it gives three distinct layers.
Since, the density of organic solvent mixture is higher than
water, it forms a lower layer (which contains denatured

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 proteins) while the upper layer is aqueous in nature and
contains nucleic acids. Chloroform causes denaturation of
proteins while isoamyl alcohol prevents the formation of
foam and helps in stabilising the interface between lower
organic and upper aqueous phase, that can be separated
by pipetting. The nucleic acids from aqueous phase are
precipitated by addition of ethanol to aqueous medium
that reduces its polarity, which makes aqueous medium
as non-polar and thus nucleic acids become insoluble that
were otherwise soluble in aqueous medium. To remove
RNA, the enzyme ribonuclease A is added that digests RNA
into ribonucleotides. DNA is then isolated by centrifugation
and stored at low temperatures (Fig. 3.1).

RNA Isolation
RNA is single stranded, while DNA is mostly double
stranded. Ribonucleases (RNases), a group of enzymes that
degrade RNA molecules, are abundant in the environment
and it is difficult to
Cell lysis and dissolution remove or destroy RNases
Cell lysis can be achieved using buffers or reagents containing completely. Thus, it is
chaotropic agent Guanidinium isothiocyanate (GITC). often difficult to isolate
intact RNA.
Denaturation of DNA and proteins
Total RNA is extracted
DNase can be used to degrade DNA, while proteinase K can be
frombiologicalsamplesby
added to digest proteins. Alternatively, repeated organic extraction
using phenol and chloroform or dissolving the sample in buffers using a specific reagent
containing guanidinium salts can also be used to remove proteins. known as guanidinium
isothiocyanate (GITC)-
Denaturation and inactivation of RNases phenol-chloroform. GITC
This can be achieved using any of the chaotropic agents, such is a chaotropic reagent
as phenol and chloroform. and acidic in nature as
it disrupts the hydrogen
Separation of cellular components bond and releases energy
RNA can be separated from other cellular components by adding to increase entropy
chloroform and centrifuging the solution. This separates the
(chaos) that reduces
solution into two phases: organic and aqueous phases. The
aqueous phase contains RNA. hydrophobic effect of
the solution resulting
in the aggregation of
Precipitation
proteins and nucleic
RNA is often recovered from the aqueous phase using isopropanol.
acids. Phenol causes
denaturation of proteins
Fig. 3.2: (a) Flow chart for the isolation of RNA

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 whereas chloroform solubilises lipids. Chloroform also
enhances specific gravity of phenol with respect to water.
When biological samples are treated with GITC solution and
subjected to centrifugation, the solution gets separated into
three phases: upper aqueous phase, followed by interface
and organic phase. Although total RNA is extracted in the
aqueous phase due to the acidic nature of the reagent
whereas DNA and denatured proteins are retained in the
interface or organic phase. This step also inactivates RNase
enzyme that may hydrolyse RNA. Subsequently, RNA from
aqueous phase is precipitated with the help of isopropanol
[Fig. 3.2 (a) and b)].

Guanidinium isothiocyanate (GITC)-

phenol-chloroform
Sample preparation
by homogenisation or lysis

Aqueous phase
(contains RNA)

Phase Collect aqueous phase

]
separation in sep a rate tube RNA
and add isopropanol Precipitation

Protein Using
Interface isopropanol
DNA (Contains precipitated
proteins)
RNA

Organic phase
(Contains DNA, lipids and proteins) RNA pellet

Fig. 3.2: (b) RNA extraction

Box 1: Separation of Plasmid DNA from Genomic DNA

Two types of DNA molecules are isolated in cloning experiments. One is plasmid DNA and
the other genomic DNA from bacteria. Chromosomal DNA is separated from plasmid DNA by
boiling bacterial lysate. The boiling of lysate causes irreversible denaturation of chromosomal
DNA and denaturation of proteins including deoxyribonuclease. Boiling causes formation of a
gel, which is precipitated by centrifugation. On the contrary, partially denatured plasmid DNA
(due to boiling) is renatured as circular double helix and become soluble. In another method,
bacterial suspension is lysed and its contents are denatured by anionic detergent SDS and
NaOH solution. The broken cell wall, chromosomal DNA and denatured proteins are clumped
as a large mass coated with SDS that are precipitated from solution by replacing sodium
ions (Na+) with potassium ions (K+). The precipitate is then separated by centrifugation. The
plasmid DNA is isolated from the supernatant by ethanol precipitation.

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 3.3 Enzymes used for Recombinant DNA
Technology
Enzymes constitute an important tool in rDNA technology.
The major task of the manipulation of the DNA involves
cutting and ligation of the vector DNA and the gene of
interest. For this, the natural abilities of different enzymes
found in organisms are exploited. The major enzymes used
in the rDNA technology are:
(i) Nucleases: Nucleases are the enzymes, which cleave
nucleic acids by hydrolysing the phosphodiester
bond that joins the sugar residues of adjacent
nucleotides. Some nucleases are DNA specific
called DNases and some are RNA specific called
RNases. There are two major types of nuclease
enzymes depending on their preference of the
location of phosphodiester bonds of polynucleotide
chains (DNA or RNA or synthetic polynucleotide
chain) namely, exonuclease and endonuclease.
Exonuclease, as the name suggests, removes the
nucleotides one at a time, i.e., mononucleotides,
(a) An exonuclease

Cleavage Cleavage
Nucleotide Hydrogen bond
5' 3'
3' 5'

Phosphodiester bond

5' P
3' OH

(b) An endonuclease

Cleavage
Cleavage

Blunt end Sticky end

Fig. 3.3: (a) An exonuclease, which removes nucleotides from the end of DNA molecule
(b) An endonuclease, which breaks internal phosphodiester bonds

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 either from the 3′ or 5′ ends of polynucleotide
chains. Endonuclease, on the contrary, breaks
internal phosphodiester bonds within a DNA or
RNA molecule [Fig. 3.3 (a) and (b)].
(ii) Restriction endonuclease/enzyme (RE):

Endonuclease enzymes that cleave DNA molecules

at a specific position are called restriction
endonucleases or restriction enzymes. They Box 2
are mostly found in bacteria and archaea that The 1978 Nobel Prize in
provide a defense mechanism against invading Physiology or Medicine
bacteriophages. RE recognises and binds to a was awarded jointly to
specific DNA sequence called recognition sequence Werner Arber, Daniel
or site, often consisting of 4 to 8 bp. Nathans and Hamilton
Restriction enzymes are categorised mainly into three Smith for the discovery
of ‘restriction enzymes’
groups (Type I, II and III) based on their co-factor
and their application to
requirement and the position of their DNA cleavage site the problems of molecular
relative to the target sequence. Type I enzymes cleave genetics. HindII was the
DNA at a site that is about 1000 bp from the recognition first restriction enzyme to
site and require S-adenosyl methionine (SAM), Mg2+, ATP be isolated by Hamilton
and has DNA strand cleavage, methylase and ATPase Smith.
activities. Type II enzymes cleave within the recognition
site and require Mg2+ and has only DNA strand cleavage
activity (Fig. 3.4). Type II REs find application in rDNA
technology. Type III enzymes cleave at sites about 24 to
26 bp away from the recognition site; require S-adenosyl
methionine (SAM), Mg2+, ATP and has DNA strand
cleavage and methylase activities (Table 3.1).
Table 3.1: Types of Restriction Enzymes

Cleavage site Endonuclease and methylase function Examples

Type I Random around 1000 Endonuclease and methylase function EcoKI
bp away from the on a single protein molecule EcoAI
recognition site
CfrAI
Type II Specific within the Endonuclease and methylase are EcoRI
recognition site separate entities BamHI
HindIII
Type III Random 24–26 bp away Endonuclease and methylase function EcoP1
from the recognition site on a single protein molecule HinfIII
EcoP15I

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 The recognition sequences of widely applied Type II REs
are palindromic sequences, meaning the sequence on
the forward direction on a double stranded DNA reads
same in a reverse direction on the complementary strand.
These enzymes break specific phosphodiester bond in
both strands of the DNA molecule within the restriction
sequence recognised by the enzyme or at the site or near
the sequence. It generates a 5′-phosphate group at one end
of the break and a 3′-hydroxyl group at the other end of the
break (Fig. 3.4). Several REs cleave at different locations on
the two DNA strands to produce staggered cut having short
single-stranded protruding ends called cohesive or sticky
ends. Some REs produce blunt ended cut by cleaving both
strands of DNA at same location (Fig. 3.4).

5 'P ' P A G C T T OH 3'

A A G C T T OH ' ' P A OH 3'
3 5 5
HindIII
' OH T T C G A P ' 3'
OH A P '
5 5
OH T T C G A A P 5 '
3' 3
Digest
5' Sticky ends

' P C T G C A G OH 3'
5 ' P C T G C A OH 3'
5
' P G OH 3'
5

PstI 3'
OH G P '
5
3'
OH
G A C G T C P 5' 3'
OH A C G T C P '
5

Digest
3' Sticky ends

5 ' P G A T A T C OH 3' ' P G A T OH 3'

5 5 ' P A T C OH 3'
EcoRV
OH T A G P '
3' 5
3 ' OH C T A T A G P 5' 3'
OH C T A P '
5

Digest
Blunt ends

Fig. 3.4: Type II REs generating sticky or blunt ends

Let us now understand the nomenclature of restriction

enzymes. The enzyme is named after the microorganism
from which it is isolated. The first capital letter represents
the genus, the second and third letters represent species.
The fourth letter specifies the strain of the microorganism.
And the last Roman number represents the number of the
enzyme isolated from the species (Table 3.2).

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 Table 3.2: Nomenclature for restriction endonucleases

EcoRI
Escherichia (E) genus
coli (co) specific epithet
strain Ry13 (R) strain
first endonuclease (I) order of identification
HindIII
Haemophilus (H) genus
influenzae (in) specific epithet
strain Rd (d) strain
third endonuclease (III) order of identification

(iii) DNA ligase: Ligase enzyme facilitate the joining of

DNA strands together by catalysing the formation of
a phosphodiester bond in the duplex form (Fig. 3.5).
Bacterial DNA ligases, from E. coli ,use the hydrolysis
of NAD as their energy source, whereas ATP is the
energy source for DNA ligases from bacteriophages
(e.g., T4) and eukaryotic cells. The 5′ P(5′ – PO4)
group of one chain makes a covalent linkage with
the 3′-OH group of adjacent chain. T4 DNA ligase
is used to join two DNA molecules having cohesive
ends or blunt ends. E. coli DNA ligase is used to
join cohesive ends.

ATP (or NAD+)

(a) 5 3 Ligase 5 3
A T G C A T G C
T A C G T A C G
HO AMP
3 P 5 + 3 5
PPi
(b)

Sticky/cohesive end ligation

(c)

Blunt end ligation

Fig. 3.5: Ligation of DNA by ligase (a) Formation of phosphodiester bond (b) Ligation of sticky end
(c) Ligation of blunt end

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 (iv) DNA polymerases: DNA polymerases are a group
of polymerases that catalyse the synthesis of new
DNA strand by using mono-deoxyribonucleoside
triphosphates (dNTPs) on a template strand. A DNA
polymerase enzyme synthesises new DNA strand in
5′→3′ direction (Fig. 3.6). It cannot initiate synthesis
of a new DNA strand. In addition to dNTPs, they
require a primer (oligonucleotide) carrying a free
3′-end hydroxyl group that can be used as the
starting point of chain growth. DNA polymerase I
of E. coli exhibit several other activities, such as
5′→ 3′ exonuclease and 3′→ 5′-exonuclease.
New strand Template 5'
5'
strand
3'
3'
Sugar A T A T

Phosphate

C G C G

G C G C
3'
OH
T A T
3'
OH
A

P C C
P P Pi
P OH
Pyrophosphate
5' released 5'
Nucleoside triphosphate

Fig. 3.6: DNA polymerase adds nucleotides at 3′OH end of the DNA molecule

(v) Alkaline phosphatase: Alkaline phosphatase is

used to remove the terminal phosphate group from
5′ end of DNA strands.
(vi) Polynucleotide kinase: Using polynucleotide
kinase, a phosphate group can be attached to
hydroxyl (-OH) group present on 5′ end of DNA.
Polynucleotide kinase has the reverse effect of
alkaline phophatase, adding phosphate groups
onto free 5′ termini.

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 (vii) Terminal deoxynucleotidyl transferase or
terminal transferase: This enzyme can add
similar nucleotide residues to form a homopolymer
tail on 3′ end of a DNA strand. Unlike most DNA
polymerases, it does not require a template.
(viii) Reverse transcriptase: It is also called RNA
directed DNA polymerase and is found in many
retroviruses. It is used to generate complementary
DNA (cDNA) strand from a–RNA template, a process
termed as reverse transcription (Fig. 3.7).
Reverse
transcriptase
dNTPs
RNA
5' 3'

Primer

5'

RNA-dependent DNA polymerase

(Reverse transcriptase) activity

RNA Strand
5' 3'

3' 5'

cDNA Strand

Fig. 3.7: Reverse transcription

(ix) Poly A polymerase: It incorporates adenine

residues to hydroxyl group of 3′ end of RNA
(Fig. 3.8).

Poly A polymerase A A

A A
A
5' 3'
dATPs
mRNA

5' AAAAAn 3'

mRNA

Fig. 3.8: Addition of dATPs by poly A polymerase

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 3.4 Modes of DNA Transfer
Transfer of a foreign DNA molecule to a host cell (prokaryotic
or eukaryotic) from its surrounding environment is one of
the basic steps in rDNA technology. In nature, bacteria
obtain foreign DNA molecules from its surroundings in
three different ways, which are: (i) transformation, (ii)
transduction and (iii) conjugation.
(i) Transformation: Transformation is genetic
alteration of a cell resulting from the direct uptake
of exogenous DNA molecule from its surroundings
through the cell membrane and gets incorporated
in the recipient genetic material. Recipient cells
with foreign DNA molecule are referred to as
transformants (Fig. 3.9). Transformation occurs
naturally in some species of bacteria.

(a) Transformation with (b) Transformation

DNA fragment with plasmid

uptake of DNA uptake of plasmid

Fig. 3.9: Transformation in bacteria

(ii) Transduction: Viruses may also mediate the

uptake of foreign DNA into the genome of a cell.
Viruses that specifically infect bacterial cells are
known as bacteriophages. Bacteriophages on
infecting follow a lytic cycle or a lysogenic life cycle
in the host. In lysogenic life cycle, the
bacteriophage genome gets incorporated into
bacterial DNA, and remains dormant for several
generations. After a period of time when phage
genome gets excised from the host DNA, they
occasionally take small sequences of bacterial
DNA with them. Phage genome containing bacterial
DNA is then packaged into phage coat proteins to

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 form a complete, recombinant viral particle. When
this phage infects a bacterial cell, the recombinant
phage genome containing bacterial DNA is
introduced into bacteria (Fig. 3.10). The recipient
bacterial cell is referred to as transductants.

Virulent Phage

Phage DNA

Generalised Transduction Specialised Transduction

Phage DNA integrates

into bacterial DNA
(Lysogenic life cycle)

Cell Lysis
(Lytic life cycle)

Cell Lysis

Fig. 3.10: Transduction in bacteria

(iii) Conjugation: Conjugation is referred to as transfer

of genetic material (DNA) from one bacterium to
another through cell-to-cell direct contact. The
bacterial cell that transfers its DNA is called
the donor cell and the one that receives is the
recipient cell. Conjugation is usually mediated by

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 Conjugation pilus F plasmids that carry a DNA sequence
encoding for the fertility factor, or F–
F plasmid Bacterial chromosome factor. The F-factor forms a thin tube-
like structure called pilus, through
which the donor cell makes contact with
the recipient. A nick is made in one of the
F + Cell F - Cell
strands of double stranded F-plasmid
by an enzyme relaxase in the donor cell
and this strand is transferred to the
recipient cell through pilus. Inside both
donor and recipient cells, the single-
stranded DNA undergoes replication
to form double-stranded F plasmid
identical to the original F plasmid (Fig.
3.11).
F + Cell F + Cell
In rDNA technology, the rDNA is
introduced (transferred) in host cells by
Fig. 3.11: Bacterial conjugation numerous methods. Chemical (calcium
chloride, lipofection, etc.) and physical
(electroporation, microinjection and gene gun) methods
for introducing foreign DNA molecules into host cells are
commonly used. In calcium chloride method, the DNA to
be transferred is mixed in a solution containing positively
charged calcium and the negatively charged group of DNA
to form a complex. The host cells take up the foreign DNA
molecule by a process of heat shock. In electroporation
method, transient micropores are created on the membrane
of host cells by exposing them to mild electric current in
the presence of foreign DNA molecules. The recombinant
DNA molecules enter into host cells through transient
micropores. Lipofection (or liposome transfection) is a
technique used to inject genetic material into a cell by
means of liposomes, which are vesicles that can easily
merge with the cell membrane since they are both made
of a phospholipid bilayer. Foreign DNA molecules can
be introduced directly to the nucleus of host cells using
specialised automated Microinjection apparatus. In
biolistic method, with the help of a gene gun (particle
gun), microscopic particles (gold, nickel, tungsten) coated
with foreign DNA are bombarded to cells at high velocity so
that foreign DNA molecule enters inside the cell (Fig. 3.12).

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 (a) DNA
++
Ca

++ ++
Ca Ca 42C
++
Ca Ca
++ ++ ++
Ca Outside Ca
++
Outside Ca
- - - - -- - -- - - - - -- - -- - - - - -- - -- - - - - -- - --
Adhesion Adhesion
zone
zone Heat ++
Ca
++ Ca
- --- ---- - - --- ---- - shock - --- ---- - 0C - --- ---- -
Lipid Inside Cell 42C Lipid Cell
Ca
++ Inside ++
molecules membrane molecules Ca membrane

Electric pulse induces

Cell ‘heals’ with
(b) Cell a voltage across
Gene of gene inside
membrane cell membrane
Interest

Before pulse During pulse After pulse

(c)
Fusion Cell membrane
DNA (Phospholipid bilayer)
+
Cell
Hollow
Liposomes
DNA to
be delivered
Outside Inside
Phospholipid
bilayer
Fused liposome
to recipient cell

(d) (e)
Injection
Gold microparticles
Projection
coated with DNA
gun

Fig. 3.12:  Methods of DNA transfer (a) Chemical (CaCl2 ), (b) Electroporation, (c) Lipofection
(d) Microinjection and (e) Biolistic method

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 3.5 Screening and Selection
Selection of transformed bacteria with recombinant
vectors is the most essential step for a successful cloning
experiment. Here, the objective is to identify the transformed
cells having recombinant vector from a mixture of
non-transformed cells. Success rate of insertion of an insert
into a plasmid and subsequent transfer of recombinant
plasmids to bacteria is very low. Therefore, it is difficult to
select a few bacteria containing plasmids with insert from
a large number of bacterial populations without the insert.
The method of selection of recombinant cells is based on
the principle of difference in biological traits present in hosts
with recombinant DNA from those without recombinant
DNA. Thus, the recombinant cells are distinguished
from non-recombinants based on their expression or
non-expression of certain traits, such as antibiotic
resistance, or expression of some specific proteins, such
as β-galactosidase or Green Fluorescent Protein (GFP), or
dependence/independence of a nutritional requirement,
such as amino acid leucine. On the basis of this principle,
the selection procedure can be divided into two main types
as described in the following section.
(i) Direct selection of recombinants: In this method
of selection, transformed cells are distinguished
from non-transformed cells based on the expression
of certain traits. For example, bacterial cells (host)
are not resistant to a particular antibiotic but when
they take up the plasmids containing antibiotic
resistant gene, they become resistant to that
specific antibiotic. These cells will survive and grow
in a media containing the antibiotic(s), whereas the
host cells without plasmid will be killed when they
are exposed to antibiotics.
(ii) Selection of recombinants by insertional
inactivation: This is more efficient than the direct
selection method. In this method, a vector having
two markers (either two antibiotic resistant genes,
or one antibiotic resistant gene and one lacZ
gene) is used. When the gene of interest (insert
DNA) is inserted into one of the selection marker
genes in the vector, its expression is disrupted
and hence called insertional inactivation. Let

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 us use a plasmid with two antibiotic resistance
genes—one for ampicillin (ampR gene) and the
other for tetracycline (tetR gene). The target DNA
(insert) is inserted into ampR gene of the plasmid
making them recombinant plasmids. Now we
have plasmids with insert and without insert.
When this plasmid mixture is added to a culture
of bacteria as described earlier, there will be
three different populations of host bacterial cells:
(i) host cells without plasmids (non-transformed),
(ii) transformed host cells with plasmids without
insert and (iii) transformed host cells with
recombinant plasmids (with insert). Now it is
essential to identify those cells that have received
the recombinant plasmid. This process of
screening is based on the property of resistance
to ampicillin, which is lost in the host cell having
recombinant plasmids. The insert gets cloned
in ampR gene leading to insertional inactivation
of ampicillin resistance gene (ampR) (Fig. 3.13).
When these bacteria are plated on a media
containing tetracycline, the non-transformed
cells get eliminated as they are sensitive to it.
Only transformed cells (with functional tetR)
multiply and form colonies as they are resistant
to it. There will be two types of colonies (master
plate)— one of transformed cells having plasmid
without insert (non-recombinant) and the other
of transformed cells having plasmid with insert
(recombinant) (Fig. 3.13). By using nitrocellulose
membrane, bacterial cells from the master plate
colonies are plated on a solid media containing
ampicillin. Transformed cells with vectors (without
insert) will only multiply to form colonies (replica
plate) while transformed cells with recombinant
vectors will not grow because their ampR gene has
been inactivated. Now, if we compare the master
plate with replica plate, the colonies present
in master plate and absent in replica plate are
the transformed cells with recombinant vector
containing DNA insert of interest (Fig. 3.13).

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 Foreign
Tetracycline DNA inserted Foreign DNA inserted into
resistant gene ampicillin resistance gene

Ampicillin Recombinant
resistance gene plasmid

Mixture of non-recombinant Bacterial host cell

and recombinant plasmid

Transformed bacterial Non-transformed

cell with non-recombinant plasmid bacterial cell Transformed bacterial
cell with recombinant plasmid

Tetracycline in media
Colonies with recombinant/
non-recombinant plasmid
Non-transformed cells
are not resistant to tetracycline
and do not form colonies on
Colonies with media containing tetracycline
recombinant plasmids

NITROCELLULOSE
MEMBRANE
Transformed bacterial cells
Ampicillin that took up non-recombinant
Transformed bacterial
in media plasmids formed colonies
colonies with recombinant/
non-recombinant plasmid

Tetracycline in media

REPLICA PLATE MASTER PLATE

Bacterial cell that took up recombinant Transformed bacterial cell colonies

plasmids are sensitive to ampicillin and do not in tetracycline media
form colonies on media containg ampicillin

Fig. 3.13: Selection of recombinants by insertional inactivation

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 Restriction site

lacZ gene

Ampicillin
ampR
resistance gene
Foreign DNA Plasmid vector

Restriction digestion of vector

and foreign DNA

Sticky
ends

R
amp

amp R Recombinant and

ampR non-recombinant
Plasmid

R
ampR
amp
ampR

Transformation
AMP

Bacterium that does Bacterium takes up Bacterium takes up

not take up plasmid is non-recombinant plasmid recombinant plasmid, cannot
not ampicillin-resistant with intact lacZ gene produce β-galactosidase enzyme

Blue colonies have White colonies

non-recombinant have recombinant
plasmids plasmids

Fig. 3.14: Blue-white selection method

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 Blue-white selection method is another example of
insertional inactivation selection method to select the
recombinant transformed cells. In this method, lacZ gene
present in plasmid vector (refer to Vector section of Chapter
2) expresses the β-galactosidase enzyme. β-galactosidase
cleaves a colourless chromogenic, substrate called X-gal
(5  
Bromo-4-chloro-3 indolyl-beta D-galactoside), an
analog of lactose to form 5-bromo-4-chloro-indoxyl, which
spontaneously dimerises to produce an insoluble blue
pigment called 5,5′-dibromo-4,4′-dichloro-indigo. When lacZ
gene in the plasmid is inactivated due to insertion of the insert
DNA, then the enzyme β-galactosidase is not expressed in
hosts containing recombinant plasmids (Fig. 3.14).
During transformation experiment, the bacterial
cells (both transformed and non-transformed) are plated
on an ampicillin and X-gal-IPTG (Isopropyl β-D-1-
thiogalactopyranoside) containing solid media. The non-
transformed cells get eliminated and only the transformed
cells multiply and form colonies. Two types of colonies will
be formed i.e., blue colour and white colour colonies. The
bacterial cells in blue colonies contain a vector with an
uninterrupted lacZ, (no insert) while cells in white colonies,
where X-gal is not hydrolysed, indicate the presence of an
insert in lacZ, which disrupts the formation of an active
β-galactosidase.
Alternative methods have been developed in order to
screen transformed bacteria, e.g., Green Fluorescent
Protein (GFP). The concept is similar to lacZ in which
a DNA insert can disrupt the coding sequence within a
vector and thus disrupt the GFP production resulting in
non-fluorescing bacteria.

3.6 Blotting Techniques

Blotting techniques are widely used by scientists to
separate and identify DNA, RNA and proteins from a
mixture of molecules. This technique immobilises the
molecule of interest on a support, which is a nitrocellulose
or nylon or polyvinylidene difluoride (PVDF) membrane.
It uses hybridisation techniques for the identification of
specific nucleic acids and genes. Both nitrocellulose and
PVDF membranes are highly hydrophobic and chemically

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 resistant to a broad range of chemicals. They have high
affinity for binding to proteins and nucleic acids. Once
proteins or nucleic acids are transferred to membranes,
they are immobilised on the membrane. A specific protein
can be detected on the membrane by using its specific
antibody. Similarly, by using a specific nucleic acid probe,
one can detect the desired nucleic acid on the membrane by
hybridisation. Detection methods used in blotting techniques
are chromogenic, fluorescence, chemiluminescence or
radioactive. There are mainly three types of blotting
techniques used in biotechnology—southern blotting,
northern blotting and western blotting.
Southern blot technique: The original blotting technique
was invented by British biologist Edwin Southern as
a method to detect specific sequence in DNA samples.
In Southern blotting, large DNA molecules are cut into
small pieces by restriction endonuclease. The DNA
fragments are separated on agar gel based on their size
by electrophoresis. DNA from the gel is transferred on to
nitrocellulose membrane through capillary action. For
this, a solid support is placed in a tray. Buffer solution is
added in the tray to half the height of the solid support. A
Whatman paper strip is placed on the solid support that
touch the buffer on two sides. The gel having DNA is kept
DNA
Paper towels
-
Migration Filter papers
Nitrocellulose membrane
Gel
Electrophoresis +
Whatman
Buffer solution
Solution passes through
gel and

Probe hybridi sed

to complementary
sequence Gel
Membrane

Expose Remove DNA transferred

X-ray m unbound
Autoradiogram Wrap in to
probe
cling m Hybridise with
unique radioactive
probe

Fig. 3.15: Identification of desired DNA by Southern blotting

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 on this Whatman strip. A sheet of nylon or nitrocellulose
membrane is placed on the top of the gel. Pressure is
applied evenly on the gel by placing a stack of filter papers
or paper towels and a weight on top of the membrane and
gel. Buffer solution by capillary action moves through the
gel and membrane onto filter papers. Along with buffer
solution, DNA moves to the positively charged membrane.
The membranes after transfer of nucleic acids, serve
as the replica of their respective gels. The membrane is
then baked to permanently attach or fix the transferred
DNA to the membrane which is mixed with probes. The
blot membrane is then washed to remove unhybridised
probes. The desired DNA sequence on the membrane is
subsequently detected using probe (Fig. 3.15). Probe
is a single DNA strand, complementary to the sequence
present in the DNA fragment to be identified. The probe is
labeled with a detecting tag which may be of radioactivity,
fluorescence or chemical nature. The labeled DNA probe
anneals with its complementary strand in the membrane.
Location of the target DNA fragment is identified by
visualisation on X-ray film by autoradiography.

Northern blotting technique: It is used to detect specific

RNA molecules in a mixture of RNA. It was developed by
American scientists J. Alwine, David Kemp and George
Stark in 1977. Like Southern blotting, it starts with the
extraction of total RNA from a homogenised tissue sample
or from cells. They are separated on a agarose gel based on
their size by electrophoresis. Then they are transferred to
a membrane where they are immobilised. A nylon
membrane with a positive charge is most effective for use
in northern blotting since the negatively charged nucleic
acids have a high affinity for them. The transfer buffer
used for the blotting usually contains formamide because
it lowers the annealing temperature of the probe-RNA
interaction, thus eliminating the need for high temperatures,
which could cause RNA degradation. Once the RNA has
been transferred to the membrane, it is immobilised
through covalent linkage to the membrane by UV light or
heat. It is then mixed with radioactive probes. The probes
are specifically designed for the RNA of interest, so that
they will hybridise with RNA sequences on the blot

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 corresponding to the sequence of interest. The blot is now
washed to remove extra probes. The labeled probe is then
detected by autoradiography, which appears as dark bands
on X-ray film or by fluorescent labels (Fig. 3.16).
RNA separated
RNA by size
Extraction Electrophoresis

Sample
Northern blotting
(transfer of RNA
to nylon membrane)
Labeled
probes

RNA xed to
Visualisation membrane with
labeled RNA on UV or heat
X-ray lm
Membrane hybridised
with labeled probes

Fig. 3.16: Identification of desired RNA by Northern blotting

Western blotting technique: The name was coined by W.

Neal Burnette in 1981. It is a technique used to detect
specific proteins in a sample of tissue homogenate or
extract. Proteins are isolated from a source. They are
separated on SDS-PAGE gel based on their electrophoretic
mobility, which depends on charge, molecule size and
Labeled antibody
Protein Separated Electro transfer of treatment
samples proteins proteins from gel to
membrane (Electro transfer)

SDS-PAGE

Develop

Protein bands visualised Autoradiography

Fig. 3.17: Identification of desired protein by Western blotting

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 structure of the proteins. They are transferred to a
nitrocellulose membrane. The desired protein is detected
on membranes using an antibody specific to the protein
(Fig. 3.17).
Using all the three blotting techniques, a person can
identify a gene and its expression. For example, a gene in a
DNA sequence can be identified by Southern blotting, and
its transcripts (RNA) can be identified by northern blotting,
and finally the expression of a protein from mRNA (by
translation) by western blotting.

Box 3: Eastern Blot

Eastern blot is used for the detection of specific post-translational modification of proteins.
Proteins are separated by gel electrophoresis before being transferred to a blotting matrix
where upon post-translational modifications are detected by specific substrates (cholera
toxin, concanavalin, phosphomolybdate, etc.) or antibodies

3.7 Polymerase Chain Reaction (PCR)

Several molecular and genetic experiments require
significant amount of DNA. In order to generate multiple
copies of DNA from a few copies, a technique was developed
by Kary B. Mullis, which is known as ‘Polymerase Chain
Reaction (PCR)’. In this technique, a very small amount of
DNA can be exponentially amplified to generate thousands
to millions of copies. PCR, sometimes called ‘molecular
photocopying’, is often heralded as one of the most
important scientific advances in molecular biology that
revolutionised the study of DNA to such an extent that its
inventor, Kary B. Mullis was awarded the Nobel Prize for
Chemistry in 1993.
PCR technique is based on the principle that cells use to
replicate its DNA. As the name implies, it is a chain reaction
carried out in repeated cycles, which involves the process of
heating and cooling called thermal cycling carried out by a
machine called thermocycler. It requires a heat stable DNA
polymerase enzyme that can make new strands of DNA on
template strands at a high temperature of about 72 to 78°C
(a temperature at which a human or E. coli DNA polymerase
would be non-functional). DNA polymerase typically used in
PCR is called Taq polymerase, an enzyme isolated from the

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 C G

A T
DNA
Sample Primers Nucleotides

5' 3'
Mix Buffer Taq PCR Tube
Target polymerase
sequence

3' 5'
Genomic DNA

5' Thermocycler
3'

Denaturation: Heat
1 at 95°C to separate
DNA strands

3' 5'

5' 5' 3'

Reverse
Primer 3' Annealing: Lowering the
Cycle 1 yields
2 molecules 3'
2 temperature to 50–55°C to form
Forward hydrogen bond of primer with
5' Primer 5' target sequence
3'

5' 3'
5' 3'
Extension: Taq DNA
3 polymerase adds
nucleotides to the 3' end
of each primer at 72°C
5' 3' 5'
3'

5' 5' 3' 3'

3' 3' 5'
5'
Cycle 2 yields
4 molecules
5'
3' 5' 3'
3' 5' 3' 5'

3'
3' 5' 3' 5'
5' 3' 3' 5' 3' 5' 3' 5' 3'
Cycle 3 yields 5' 5'
8 molecules;
2 molecules
(in white boxes) 5'
3' 5' 5' 3' 5' 3' 5'
match target 3' 3' 5' 3'
5' 5' 3'
sequence 3'

Fig. 3.18: Steps of polymerase chain reaction

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 thermophilic bacterium, Thermus aquaticus, which inhabits
hot springs. Another enzyme Pfu polymerase isolated from
Pyrococcus furiosus is used widely because of its higher
fidelity when copying DNA. Like other DNA polymerases,
Taq polymerase also requires a primer, a short sequence of
nucleotides that provides 3′-OH end to start DNA synthesis.
Two types of single stranded synthetic deoxyoligonucleotide
primers (called forward and reverse primers) are used in
each PCR reaction that is complementary to the DNA
sequences in the template strands flanking the target region
(region that should be copied). They are designed from the
pre-existing knowledge of the sequence of DNA template to
be amplified.
PCR involves three steps — denaturation, annealing
and extension (Fig. 3.18). The first step, i.e., denaturation
is accomplished by heating the double stranded DNA to
be amplified to a temperature of about 94–95°C. At high
temperature, hydrogen bonds that hold two complementary
strands of DNA molecule break down and each strand serves
as template for the synthesis of its new complementary
strands. The second step is annealing during which the
temperature is lowered to around 50–55°C so that the
specific primers can anneal to their respective template
strands at their complementary sites and serve as the
starting point for copying. Lowering of temperature depends
upon the length of the primer and sequence of the primer.
In the third step, i.e., extension, the temperature is raised
to about 72°C, and the heat stable DNA polymerase begins
adding deoxyribonucleotides (dNTPs – dATP, dTTP, dCTP
and dGTP) onto the 3′-OH ends of the annealed primers.
Thus, a new chain of DNA grows from 5′ to 3′ direction
on each template. Copies of DNA strands formed by PCR
are known as amplicons. At the end of the cycle, again
the temperature is raised and the process is repeated. The
number of DNA copies doubles after each cycle hence the
number of copies at the end of each cycle would be 2n (where
‘n’ is the cycle number). Usually, 25 to 30 cycles are carried
out in a typical PCR reaction.
In PCR, the amplified product is analysed by gel
electrophoresis at the end of reaction (end point analysis).
The amount of DNA in the band of gel plate is then estimated
by measuring the intensity of the band by computer

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 Cells or tissue

Isolate total mRNA

Anneal anchored oligo (dT) primers,

mRNA AA AAA
TTTTT
First strand synthesis

mRNA AA AAA
cDNA TTTTT

RNA removal and second

strand cDNA synthesis

ds DNA

Fig. 3.19: Steps of RT-PCR.

programs and transferred into a quantitative data. This is

called semi-quantitative PCR. If DNA material is formed
from mRNA by reverse transcriptase and used in PCR for
amplification (Fig. 3.19), the method is known as reverse
transcription PCR (RT-PCR).
The latest advancement in PCR technology is
real-time quantitative PCR (real-time qPCR). In this method,

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 fluorescent markers are used, which have specific binding
affinity to double stranded DNA. When bound to dsDNA,
they exhibit fluorescence behaviour. Fluorescence emission
is detected and quantitated by a detector. The amount of
fluorencence emitted is directly proportional to the amount
of double stranded PCR product. Since, the amount of PCR
product formed can be measured after each PCR cycle, and
hence, it is called real-time quantitative PCR. One of the
fluorescent dyes used in real time PCR is SYBR green. The
dye only binds to double stranded DNA.
The machine in which PCR reaction is conducted is
known as thermocycler. These are automated machines
having control points where one can set three gradients of
temperature (for denaturation, annealing and extension)
for different time periods for each step. For real-time
qPCR thermocycler it has a detector to measure emitted
fluorescence. In real-time PCR, gel electrophoresis is not
needed as in case of conventional PCR.
PCR has several applications in molecular biology and
rDNA technology. One of the applications of PCR is to
quantify mRNA to assess the expression of a gene. It is also
used to amplify minute DNA samples collected from crime
scenes and fossils for further investigation.
Box 4

Box 4

The Novel Coronavirus (nCoV) Severe Acute Respiratory Syndrome

Coronavirus-2 (SARS-CoV-2) and COVID-19 Disease
(Corona Pandemic)
The International Committee on Taxonomy of Viruses on 12 February, 2020, officially named
2019-nCoV virus as SARS-CoV-2, and on the same day, World Health Organization (WHO)
announced it to be responsible for the pandemic Coronavirus disease 2019 (COVID-19). SARS-
CoV-2 is an enveloped virus, which contains crown-like spikes on its outer surface.
The genome of SARS-CoV-2 is a single-stranded positive (sense) RNA of 30 kb with G + C
content of 38%. Two-thirds of viral RNA encode a number of non-structural proteins (NSPs), which
include papain-like protease (PLpro), 3-chymotrypsin-like protease (3CLpro), RNA-dependent RNA
polymerase (RdRp), helicase (Hel) and exonuclease (ExoN) as major proteins while the rest

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 are accessory proteins that are involved in the transcription and replication of the virus. The
rest part of the virus genome encodes four essential structural proteins, including spike (S)
glycoprotein, small envelope (E) protein, membrane (M) protein, and N phosphoprotein (N)
protein, and also several accessory proteins that interfere with the host immune response.
On the basis of the structure, the RT – PCR tests have been efficiently optimised, and
mRNA vaccines have been designed and being administered (Chapter 4).

0 5,000 10,000 15,000 20,000 25,000 30,000

ORF4
ORF1b
ORF1a ORF2 ORF9
ORF5 - 3'
5' -

PLpro 3CLpro ExoN E M U

Hel S
RdRp

Membrane protein

Envelope protein

Spike protein

Nucleocapsid protein
Enclosing RNA

Lipid membrane

Schematic diagram of the SARS-CoV-2 genome organisation and a virion. The genome contains a
5′-untranslated region (5′′- UTR), open-reading frames (ORFs) 1a and 1b encodes non-structural
proteins, 3-chymotrypsin-like protease (3CLpro), papain-like protease (PLpro), helicase (Hel), and
RNA-dependent RNA polymerase (RdRp) besides accessory proteins, The other ORFs code for
structural S protein (S), E protein (E), M protein (M), and N phosphoprotein (N).

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 Box 5: Application of RT-PCR and COVID-19 detection test

RT-PCR plays an important role in the COVID-19 detection test. It is based on the principle
of real-time reverse transcription polymerase chain reaction (rRT-PCR) test that qualitatively
detects the nucleic acid from SARS-CoV-2 in the lower and upper respiratory tract specimens
[sputum, broncho-alveolar lavage (BAL)] collected by health care staff, from individuals that
are suspected of COVID-19.
Principle of the RT-PCR test is same as described in the chapter. For testing, primer and
probes are selected from Open Reading Frame gene region (ORF1a/b) and viral nucleocapsid
region (N), or the spike protein (S) of SARS-CoV-2 genome. The kit contains primer/probe
specific for N gene, ORF1a/b gene and the human RNase P. RNA is separated and purified
from the upper and lower respiratory tract specimens is firstly converted to cDNA by reverse
transcription and then amplified in real-time PCR thermal cycler. Probes consist of a reporter
dye at 5′ and quenching dye at 3′. The fluorescent signals emitted from reporter dye are
absorbed by the quencher, so it doesn’t emit signals. During amplification, probes are allowed
to bind to templates and are cut off by Taq enzyme (5′-3′ exonuclease activity), separating
reporter dye from the quencher, and generating fluorescent signals. The PCR instrument can
then inevitably draw a real-time amplification curve that is based on the change in signal, and
finally realising the qualitative detection of SARS-CoV-2 novel coronavirus at the nucleic acid
level. Amplification plots shown in the figure signify the accumulation of the product over
the duration of the real-time PCR experiment. The fluorescent signal from individual sample
is plotted against the cycle number.
Ct value

Sample
Fluorescence

Threshold
No template
Baseline
Number of Cycles

Threshold level and Ct value on a RT-PCR

amplication curve

The threshold cycle or Ct value is the cycle number at which the fluorescence generated
within a reaction crosses the fluorescence threshold — a fluorescent signal significantly above
the background fluorescence. Ct refers to the number of cycles needed to amplify the viral
RNA to a detectable level. At the threshold cycle, a detectable amount of amplicon product has
been generated during the early exponential phase of the reaction. The Ct value is inversely
proportional to the amount of the gene of interest in the sample.

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 3.8 DNA Libraries
DNA molecules present in a genome of an organism are
very large in terms of the number of base pairs it contains.
The size of a DNA molecule present in any diploid cell from
any organ of your body has around 3×109 bp. In a genome,
gene sequences are arranged randomly and selecting or
isolating a gene of interest is a big task, especially when the
genomic sequences are not known. Also, a small portion
of genome is transcribed to give mRNA, whereas a major
portion of the genome remains untranscribed. It will be
very difficult to isolate a gene of interest or a sequence
of genome whose location and sequence is not known.
Hence, DNA libraries are constructed by collecting DNA
fragments that have been cloned into vectors so that the
specific DNA fragments of interest can be identified and
isolated for further study. There are basically two types
of DNA libraries (genomic and cDNA libraries), which are
described in the following section.

(i) Genomic DNA Library

A genomic library is a collection of clones of small fragments
of DNA that together represents the complete genome of an
organism. A population of identical vectors store DNA inserts,
each containing a different insert. In general, construction of
genomic library is done as shown in Fig. 3.20. First, genomic
DNA is isolated from the source, which is too large to be
incorporated into a vector and needs to be broken down
into desirable fragment sizes. Therefore, the genomic DNA
is digested with a restriction enzyme to cut the DNA into
fragments of a specific size. DNA fragments are then inserted
into vectors using DNA ligase to form recombinant vectors.
This generates a pool of recombinant DNA molecules.
The recombinant DNA molecules are now taken up by
host bacterial cells by transformation and then allowed to
multiply in a nutrient medium to form colonies. All host cells
containing recombinant vectors represent a genomic library.
The library created contains representative copies of all the
DNA fragments present within the genome of an organism.

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 DNA is extracted from DNA fragments
cells and digested with are inserted into
a restriction enzyme cloning vector

Selection of
Transformants Allowed Bacterial cells
to multiply are transformed
with vectors

Fig. 3.20: Construction of genomic library

Genomic library has several applications in

biotechnology. Genomic library of a species may be helpful
for complete sequencing of its genome. Also, one can search
for many genes that are not expressed in the genome of an
organism. It is also helpful in understanding the evolution
of species. Genomic library can be used to compare the
sequences of healthy and diseased tissues of the same
organism to identify genetic aberrations.

(ii) cDNA Library

Gene expression in higher eukaryotes is tissue-specific.
In specific cells, certain genes undergo moderate to high
expression. For example, the genes encoding insulin
proteins are expressed only in beta cells of pancreas while
albumin encoding genes are expressed in liver cells. Using
this information, a target gene can be cloned by isolating the
mRNA from a specific tissue. The specific DNA sequences
are synthesised as copies from mRNAs of a particular cell
type called cDNA (complementary DNA). Clones of such
DNA copies of mRNAs are called cDNA clones. The cDNA
clones of all the genes expressed in a specific cell type or
tissue of an organism represent cDNA library.
Construction of cDNA library involves the isolation of
total mRNA from a cell type or tissue of interest. mRNA
being single-stranded cannot be cloned as such and is
not a substrate for DNA ligase. It is first converted into
cDNA before insertion into a suitable vector, which can be

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 Reverse mRNA
Transcriptase 5' 3'
(RTase) RNase
3' 5'
cDNA
Source mRNA cDNA

5' 3' DNA

Polymerase
3' 5'
dsDNA

Cleaved DNA
Digestion with
restriction enzymes
Vectors

Ligation

Cleaved vectors

Insertion into
E. coli

Library amplication in rapidly

reproducing bacteria

Fig. 3.21: Construction of cDNA library

achieved using reverse transcriptase (RNA-dependent DNA

polymerase or RTase). RTase synthesise a complementary
DNA strand on mRNA by using mRNA as a template.
mRNA is then removed by RNase and the single stranded

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 cDNA is converted into double-stranded cDNA by DNA
polymerase. cDNA molecules are cloned in appropriate
host-vector system (Fig. 3.21). The total clones of cDNA
are the representative of cDNA library of the source. Since
the expressions of genes are different in different organs or
cells of an organism at different physiological states, cDNA
libraries prepared from different sources of an organism
may vary from each other.
The cDNA library has a great significance in the
applications of biotechnology. The most important
application of cDNA library is to know which genes are
active in particular tissues under a particular physiological
state. It also helps us to isolate a specific gene. Using cDNA
as probes, we can screen genomic libraries for a particular
gene.

­SUMMARY
• Isolation of nucleic acids from different organisms is the
most essential requirement for any molecular biology
experiment. There are four steps in the process of
extraction of nucleic acids, i.e., disruption of biological
samples, protection of nucleic acids from its degrading
enzymes, separation of nucleic acids from other molecules
and assessment of purity and quality of the isolated
nucleic acids.
• Various enzymes play an important role in recombinant
DNA (rDNA) technology. These are nucleases, DNA
ligase, alkaline phosphatase, polynucleotide kinase,
poly A polymerase, etc.
• The major task of the manipulation of DNA involves cutting
and ligation of the gene of interest into the vector DNA.
• Nucleases are the enzymes that cleave nucleic acids
by hydrolysing the phosphodiester bond that joins the
sugar residues of adjacent nucleotides. Two major types
of nuclease enzymes depending on its action on the
phosphodiester bonds of polynucleotide chains have been
identified, which are exonuclease and endonuclease.
• Exonuclease enzymes can remove mononucleotide either
from the 3′ or 5′ end of the DNA molecule.

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 • Endonuclease enzymes cleave DNA molecules at a specific
sequence, hence called restriction endonucleases or
restriction enzymes (REs). REs are mainly categorised
into three groups (i.e., Types I, II and III) based on their
cofactor requirement and the position of their DNA
cleavage site relative to the target sequence. Type II REs
find application in rDNA technology.
• DNA ligase can join two DNA strands together by catalysing
the formation of a phosphodiester bond in the duplex
form.
• DNA polymerases are a group of enzymes that catalyse
the synthesis of new DNA strand by using dNTPs on a
template strand.
• Alkaline phosphatase is used to remove the terminal
phosphate group from 5′ end of DNA strands.
• Reverse transcriptase is used to generate complementary
DNA (cDNA) strand from an RNA template, a process
called reverse transcription.
• In rDNA technology, the recombinant DNA is introduced
(transferred) in host cells by a number of methods,
such as chemical based transfection (calcium chloride,
lipofection etc.) and physical transfection (electroporation,
microinjection and biolistic) methods.
• Selection of transformed bacteria is the most essential
step for a successful cloning experiment, i.e., to identify
the transformed cells having recombinant vector (with
gene of interest) from a mixture of transformed and
non-transformed cells. These selection methods may be
direct or through insertional inactivation.
• In direct selection, the transformed cells are distinguished
from non-transformed cells based on expression of certain
traits, such as resistance to antibiotics.
• In insertional inactivation method, a vector is used having
two markers (either two antibiotic resistant genes or one
antibiotic resistant gene and lacZ gene).
• Blue – white selection method is another example of
insertional inactivation to select recombinant transformed
cells in which the expression of lacZ gene can directly be
observed in bacterial colonies.
• Blotting techniques are widely used to separate and identify
DNA, RNA and proteins from a mixture of molecules.
• Southern blotting technique is used to detect specific
sequence of DNA in DNA samples.

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 • Northern blotting technique is used to detect specific RNA
molecules in a mixture of RNA.
• Western blotting is used to detect specific proteins in a
sample of tissue homogenate or extract.
• Polymerase Chain Reaction (PCR) is used to amplify a
small amount of DNA into thousands to millions of copies,
which involves three steps, i.e., denaturation, annealing
and extension. The amplified product of PCR can be
analysed by gel electrophoresis at the end of reaction (end
point analysis).
• The latest advancement in PCR technology is real-time
quantitative PCR (qPCR), in which the fluorescent markers
are used that have specific binding affinity to double
stranded DNA. In qPCR, gel electrophoresis is not needed
as in the case of conventional PCR.
• DNA libraries are constructed by collecting DNA fragments
that have been cloned into vectors so that specific DNA
fragments of interest can be identified and isolated. There
are basically two types of DNA libraries — genomic and
cDNA library.
• A genomic library is a collection of clones of small fragments
of DNA that together represent complete genome of an
organism.
• The cDNA library constitutes cDNA clones of all the genes
expressed in a specific cell type or tissue of an organism.

EXERCISES
1. Describe the methods used for isolation of DNA.
2. What is the role of biological detergent in the process
of isolation of nucleic acid?
3. How does DNA isolation from plant tissue differ from
that of bacterial cell?
4. How many types of restriction enzymes (REs) are
there? Can all REs be used in rDNA technology? Give
justification.
5. What are the challenges faced during the process of
nucleic acid extraction?
6. Write the role of alkaline phosphatase, DNA ligase,
terminal transferase in rDNA technology.
7. Describe the role of chelating agent in the process of
DNA extraction.

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 8. Briefly describe the modes of DNA transfer into the
host.
9. Identify the correct statement for blue – white selection
method.
(a) A specific dye is used to stain bacterial colony.
(b) It is based on the expression of lacZ gene.
(c) The recombinant bacterial colony remains blue.
(d) lacZ gene is inserted in an antibiotic resistant
gene.
10. Identify the correctly matched pair from the following
options.
(a) Northern blot: Detect specific sequence of DNA
(b) Southern blot: Detect specific sequence of RNA
(c) Western blot: Detect specific proteins
(d) Eastern blot: Detect transcriptional modifications
in RNA
11. Identify the incorrect matched pair from the following
options.
(a) Taq polymerase: Thermus aquaticus
(b) Pfu polymerase: Pyrococcus furiosus
(c) HindIII: Haemophilus influenzae
(d) PstI: Pyrococcus stuartii
12. How are recombinants screened? Describe the methods
in detail.
13. Differentiate between the Southern, Northern and
Western blotting.
14. What is PCR? Describe in detail.
15. Write a comparative account of the genomic and cDNA
libraries.
16. Diploid human genome contains:
(a) 3.2 × 109 base pairs
(b) 6.4 × 108 base pairs
(c) 3.2 × 108 base pairs
(d) 6.4 × 109 base pairs
17. Select the incorrectly matched pair from the following.
(a) Nucleases : Hydrolyse phosphodiester bond
(b) Restriction enzymes: Cleave DNA at specific
sequence
(c) Palindromic sequence: Read same backwards and
forward
(d) EcoRI: Type I Restriction Enzyme

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 18. Assertion: PCR can be used to amplify very small
amount of DNA using DNA modifying enzymes.
Reason: PCR uses Taq Polymerase.
(a) Both assertion and reason are true and the reason
is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason
is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.
19. Assertion: Foreign gene can be introduced into
host bacterium by transformation techniques like
electroporation.
Reason: Bacteria have cell wall/membrane.
(a) Both assertion and reason are true and the reason
is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason
is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.

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