Introduction to

gene cloning
and genetic
engineering

Tools of recombinant DNA technology:

(I) Genomic DNA: The genomic DNA is isolated from a donor. The desired
gene is isolated from DNA.
(ii) Vector: The vector which is generally a plasmid which is used for the
insertion of the desired gene.
(iii) Restriction endonucleases: These are the enzymes which are used for the
fragmentation of the DNA. Endonuclease enzymes are known as molecular
scissors as they produce nick in the DNA fragment at the desired place.
(iv) Ligase: This is the enzyme which is used for the ligation (joining) of the two
fragments. The desired gene is introduced in the vector and attached with the
help of ligase enzyme.
(v) Host: The recombinant vector is introduced into a competent host cell. The
host cells are cloned or replicated for the production of more number of
recombinant vectors which can be used for the formation of product.
Restriction endonuclease/ restriction
enzyme:

Restriction enzymes are also

These enzymes make one
called "molecular scissors"
incision on each of the two
as they cleave DNA at or
strands of DNA and are also
near specific recognition
called restriction
sequences known as
endonucleases.
restriction sites.
 Restriction endonuclease:
Restriction endonuclease Cleavage site Subunits Example

Type 1 Random around Single enzyme with 3 EcoK I

1000bp away from subunits for EcoA I
recognition site recognition, cleavage CfrAI
and methylation.

Type 2 Specific within the Two different enzymes EcoR I

recognition site for modification and BamH I
cleavage. Hind III

Type 3 Random 24-26bp Single enzyme with 2 EcoP I

away from recognition subunit for cleavage Hinf III
site and recognition. EcoP15 I
• When a restriction endonuclease recognizes a sequence, it snips
through the DNA molecule by catalyzing
the hydrolysis (splitting of a chemical bond by addition of a
water molecule) of the bond between adjacent nucleotides.
• Bacteria prevent their own DNA from being degraded in this
manner by disguising their recognition sequences.
• Enzymes called methylases add methyl groups (—CH3) to
adenine or cytosine bases within the recognition sequence,
which is thus modified and protected from the endonuclease.
• The restriction enzyme and its corresponding
methylase constitute the restriction-modification system of a
bacterial species.
Difference between blunt end and sticky end
Basis of comparison Sticky end Blunt end
Formation Formed when the restriction Formed when the restriction
enzyme makes staggered cuts that enzyme makes a straight cut
are not directly opposite each through both DNA strands in the
other. same place.

Physical characteristics The ends feature overhangs that The ends are blunt and do not
allow the two ends to base-pair feature any overhangs or unpaired
and join together with another bases
DNA strand

Pairing Have unpaired DNA nucleotide on There is no unpaired DNA strand

either 5’- or 3’- strand

Also known as Cohesive ends/ Staggered ends Non-cohesive ends/ Flush ends
Exonuclease: are enzymes that work by cleaving nucleotides one at a time from the end of a
polynucleotide chain. It breaks phosphodiester bonds at either the 3’ or the 5’ end.

S1 Nuclease: is an endonuclease that specifically degrades single-stranded nucleic acids, creating a

blunt end.

DNA polymerase I: is an enzyme that participates in the process of prokaryotic DNA replication. It
synthesises DNA complementary to DNA template in 5’ to 3’ direction.

The Klenow fragment is the largest fragment that contains 5′ to 3′ polymerase and 3′ to 5′
exonuclease (proofreading) activity domains of the DNA polymerase Ⅰ. The Klenow fragment is a
large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by
the protease subtilisin.

S1 Nuclease
Reverse transcriptase (RT): also known as RNA-dependent DNA
polymerase, is a DNA polymerase enzyme that transcribes single-
stranded RNA into DNA.

Taq polymerase: is the heat-stable (thermostable) DNA

polymerase extracted from the thermophilic
bacteria Thermus aquaticus. Its predominant function is in the
polymerase chain reaction (PCR) technique, where it automates
the repetitive step of amplifying specific DNA sequences.

T4 DNA Ligase: is a ligation enzyme that can be used to join DNA

fragments.
Alkaline phosphatase: The enzyme is generally used
for the removal of single phosphate groups from 5´-
ends of linear vectors to prevent re-circularization
during cloning.

Terminal transferase: catalyses the addition of

nucleotides to the 3' terminus of a DNA molecule.

Polynucleotide kinase: They transfer the gamma

phosphate group from adenosine triphosphate
(ATP) to the 5' hydroxyl termini of DNA or RNA.
Prokaryotic host cell:
• The bacterium Escherichia coli is widely used in many
cloning protocols.
• In addition to E. coli, other bacteria may be used as hosts for
gene cloning experiments, with examples including species of
Bacillus, Pseudomonas, and Streptomyces.
• The process of transferring a DNA into a prokaryotic host cell is
called transformation.
Eukaryotic host cell:
• One disadvantage of using a prokaryotic host is that it lacks the
membrane bound nucleus (and other organelles) found in eukaryotic
cells. This means that certain eukaryotic genes may not function in a
prokaryotic host cell as they would in their normal environment,
which can hamper their isolation by selection mechanisms that
depend on gene expression.
• Also, if the production of a eukaryotic protein is the desired outcome
of a cloning experiment, it may not be easy to ensure that a
prokaryotic host produces a fully functional protein.
• Eukaryotic cells range from microbes (e.g., yeast and algae) to cells
from complex multicellular organisms (e.g., plant and animal cells).

Methods of isolation of cellular DNA and

purification

Animal
Bacterial
and plant
Steps involved

CELL LYSIS PRECIPITATION PURIFICATION

Cell Lysis

PHYSICAL CHEMICAL ENZYMATIC

Physical methods
• Homogenizer

• Sonication

• Vortex

• SDS- sodium dodecyl sulfate- is a

detergent that is known
to denature proteins (Anionic
detergent)
Chemical
method • CTAB- cetyltrimethylammonium
bromide(Cationic detergent) is a
detergent that is known
to denature proteins.
CTAB AND SDS

SDS mechanism
 Enzymatic method:
•Lysozyme: Lysozyme breaks down the bacterial cell wall by cleaving
peptidoglycan.
•Cellulase: Plant cells have cellulose in their cell wall which can be
degraded using the enzyme cellulase for isolating the genetic
material DNA.
•Chitinase: Use of chitinase enzyme, is necessary for isolation of DNA
from fungus (yeast cells).
•Proteinase K: Responsible for digestion of all the proteins in the cell
lysate.
•RNase A: It cleaves the cellular RNA (all types) which are not
required for cells.
 PRECIPITATION:

•Na+ ions: It neutralizes the negative charge

on DNA so that DNA becomes less soluble
in water and more stable.
•100% alcohol: (Isopropanol/ethanol):
White thread like structure is seen.

Purification:
• Centrifugation: DNA forms a pellet at the bottom of the
eppendorf tube.
• Suspend the pellet in 70% ethanol.
• Centrifuge the mixture.
• Collect the pellet, discard the supernatant.
• Air dry the DNA to remove ethanol.
• Add TE buffer (Tris EDTA buffer) or distilled water.
Bacterial DNA extraction and purification:
• Grow the bacterial cell in LB (Luria-Bertani) broth at 37° C in orbital
shaking incubator (150-200 RPM)
• Check OD at 600nm. If we get 0.8 to 1 (0.8 × 10^9 CFU/ml) then cells
are good to harvest.
• Harvest the bacterial cell by centrifugation 10,000 RPM for 1 min or
8000 RPM for 5 min.
• Discard the supernatant.
• To the pellet we add 100µl of PBS -Phosphate buffered saline (is
a balanced salt solution ) to wash the cell. Pipet it several times to
make a uniform suspension. It is done to separate bound and
unbound reagents.
• Centrifugation at 10,000RPM for 1 min or 8000 RPM for 5 min.

• Discard the supernatant and to the pellet add lysis buffer (Tris +
NaCl+ EDTA+ SDS) to break open the cells. We can also add
proteinase K, lysosyme and RNase.
❖Tris is used to increase permeability of cell membrane. Tris is
used to keep a stable pH. (pH 8).
❖NaCl-Sodium chloride helps to remove proteins that are bound to
the DNA.
❖EDTA-it inactivates DNase, by binding to metal cations required by
this enzyme. It inhibits divalent cation like Mg2+ and thus inhibiting
DNase.
Deoxyribonuclease (DNase, for short) refers to a group of
glycoprotein endonucleases which are enzymes that catalyze the
hydrolytic cleavage of phosphodiester linkages in the DNA backbone,
thus degrading DNA.
• Incubate the mixture for 1 hr in a water bath set to 56° C
• Add equal amount of phenol Chloroform. (It will form density gradient)
• Spin for 5 min at 10,000 RPM and collect the aqueous phase.
• To aqueous phase add 100% ethanol.
• Centrifuge at 10,000 RPM for 5 min.
• Discard the supernatant.
• To the pellet add 1 ml of 70% ice cold ethanol.
• Mix gently.
• Centrifuge at 10,000 RPM for 5 min.
• Air dry the pellet.
• Store DNA in sterilized water or TE buffer.

Vector introduction
• It is a biological tool in recombinant DNA technology.
• It is used for the delivery of desired foreign DNA into the host cell.
• A vector is a DNA molecule that have the ability to replicate
autonomously in the host cell and into which DNA fragment to be
cloned.
Properties of ideal vector:
• It should replicate autonomously.
• A vector should be less than 10 KB in size.
• It should be easy to isolate and purify.
• It should be easily introduced into the host cell.
• It should have suitable marker genes.
• Vector should consist a unique target sites and recognition sites for
various restriction enzymes.
• It should have the capability to incorporate either itself or the DNA
insert in the Genome of the host cell.

TYPES OF VECTOR

Cloning vector

Expression vector
 Cloning Vectors
Cloning vectors are small piece of DNA which have the ability and used to introduce
foreign gene of interest into the host cell.

They can be stably maintained insides the host cell.

Cloning vector are generally used to obtain multiple copies of desired foreign gene.

Example- Plasmid, Cosmid and Phages, BACs, YACs.

These type of vectors generally contains selectable marker, origin of

replication and a restriction site.

Expression Vector
Expression vector is a type of vector which not only introduces a gene of interest into the
host cell but also aids in the analysis of the foreign gene via relevant protein product
expression.

It is type of vector which is used to obtain or analyses the gene product, which may be RNA
or protein of the inserted desired gene.

Example- Only plasmid vector.

Expression vector contains enhancer, promoter region, start/stop

codon, transcription initiation, selectable marker, ori sites, and restriction site.
 Examples of some vectors and their suitable
host:
Vector Host
Plasmid Bacteria
Bacterophage Bacteria
Cosmid Bacteria
Virus (sv40, retroviruses) Animal cell
Yeast cloning vector Yeast

Plasmid Vector
• A plasmid is a naturally occurring extrachromosomal double stranded
,circular DNA.

Characteristics of Plasmid vector:

• It contains an ori site or origin of replication.
• It replicates autonomously within bacterial cell.
• It also contain selective marker such as antibiotic resistance, blue
white screening.
• Small in size (1.0 to 250 kb)
• Contains multiple cloning site.
• Easily isolated from the host cell.
 Plasmid Bolivar Rodriguez
Examples of
plasmid
vectors are: •pBR322

Plasmid University of
California
•pUC 18/19
• It is 4361 base pair long.
• This E.Coli cloning vector has the origin of replication (ori)
• It carries two sets of antibiotic resistance gene:
➢Ampicillin resistance gene( Provides resistance against
the antibiotic, ampicillin and is used as a selectable
marker.)
➢Tetracycline resistance gene ( Provides resistance against
the antibiotic, tetracycline and is used as a
selectable marker.)
• The restriction sites in pBR322 are – HindⅢ, BamHⅠ, SalⅠ,
PstⅠ, PvuⅡ, PvuⅠ, ClaⅠ and EcoRⅠ.
 PUC 18/19
• This vector is 2686 base pairs in length.
• The lacZ gene codes for ꞵ galactosidase( it will convert lactose to glucose
and galactose. This vector also encodes for ampicillin resistance.
• The restriction sites in pUC19 are – HindⅢ, BamHⅠ, SalⅠ, SmaⅠ, SacⅠ,
KpnⅠ, XbaⅠ, PstⅠ, SphⅠ and EcoRⅠ.
• pUC19 is widely used in molecular techniques due to its high copy number.
• They are smaller in size, and hence their rate of replication is high.
• This vector is also called as blue white screening vector because based on
the colour of the colonies we can make out whether they are recombinant
or non recombinant.
• We grow the bacteria in agar + X gal (5-Bromo-4-Chloro-3-Indolyl
Galactopyranoside which is a lactose analoge) ( + IPTG (isopropyl thio
galactosidase which acts as an inducer)
• If they are blue in colour then they are non recombinants.
• If they produce white colour colonies they are recombinants.

Cosmids Vector
• It is a type of hybrid plasmid.
• It contains lambda phage cos sequence.
• Cosmids = cos sites + plasmid.
• These cos sites are nothing but the cohesive ends (sticky end).
• Because of this cos sites, it's easy to pack them into phage
heads. (invitro pakaging)
• Cosmid vectors can accommodate up to 42 kb of DNA.
• If they have suitable origin of replication than they can replicate as Plasmid
within the host cells.
• It also contains selectable marker such as Ampicillin resistance gene.
• Collins and Hohn in 1978 was first to described cosmids.
 Phage (Lambda phage)
• Can incorporate larger DNA fragments which is not possible in
plasmid vector.
• This is used to clone genomic eukaryotic DNA.
• Invitro packaging is possible.
• These phage have the ability to infect the bacteria.
• Life cycle of bacteriophage contain lytic cycle and lysogenic cycle.
• In lytic cycle it is going to destroy the host organism and in
lysogenic cycle it is going to integrate into host genome.
• If we see the genome of Lambda phage, one third of the genome is
non-essential. So, we can replace this with foreign DNA.
 Phage (M13 phage)
• Is a filamentous bacteriophage.
• Single stranded circular DNA genome (6407 bp long)
• Filamentous bacteriophage which infects E.coli host through bacterial sex pilus.
• After entering into the bacterial cells, the M13 phage DNA [called +strand] is converted
into double strand molecule called replicative form.
• Replicative form contains both + and - strand that replicates until there are 100 copies in the
bacterial cell.
• Replicative form is used for genetic manipulation.
• M13 vectors are used for obtaining single stranded copies of DNA which are suited for DNA
sequencing and invitro mutagenesis.
• Upto 3 kb inserts can be cloned in M13 phage.
• The cut is made in the polylinker region that contains a series of closely spaced recognition
site for restriction enzymes. As polylinker is present in lac Z gene, recombinant cells
produces transparent plaques on X-gal agar medium.
Artificial Chromosome Vectors

There are several types of such vectors:

1. Bacterial artificial chromosomes (BAC).
2. Yeast artificial chromosomes(YAC).
3. Mammalian artificial chromosomes(MAC).
4. Human artificial chromosome (HAC).

Bacterial artificial chromosome:

• A BAC is an engineered DNA molecule (circular, double stranded), used to
clone DNA sequences in bacterial cells.
• Large fragments of DNA ranging from 100-300kb can be inserted into BAC.
Components of BAC:
➢Origin of replication (Ori), multiple cloning site (MCS), selectable marker,
PAR gene.
➢PAR gene helps in even distribution of BAC to daughter cells during
bacterial cell division.
• 1-2 vectors are found per bacterial cell. There are no gene deletion and
gene recombination or inversion in the inserted gene sequence, thus they
are more stable.
Yeast artificial chromosome:
• Are artificially constructed vector that are used for cloning DNA in yeast.
• A DNA fragment of 100-2000kb can be inserted in these vectors.
Components of YAC:
➢Two copies of yeast telomeric sequence.
➢A yeast centromere.
➢A yeast ARS (autonomously replication system, where DNA replication begins)
➢Origin of replication (bacterial origin)
➢Multiple cloning site (MCS)
➢Selectable markers (for both bacteria and yeast)
• Only one vector occurs per yeast cell.
• There are chances of gene deletion and gene recombination or inversion in the
inserted gene sequence thus they are less stable.
 Animal and plant viruses:
• CaMV: Cauliflower mosaic virus (plant virus)
• TMV: Tobacco mosaic virus )plant virus)
• Retrovirus: Moloney murine leukemia virus are the most
common retrovirus.
Other example: HIV, human T-cell lymphotropic virus type
1 (HTLV-1) causes a form of cancer called adult T-
cell leukemia (ATL)
• SV40: simian vacuolating virus 40 or simian virus 40
SV40 has been widely studied as a model eukaryotic virus

Transfer of recombinant into host cell.

(vectorless methods)
• Basic concept of transformation:
Recombinant molecules enter living cells in a process
called transformation.
• Transfection:
Transfection is a process of introducing nucleic
acid into eukaryotic cells using various chemical or physical
methods.
 Electroporation:
• The use of high-voltage electric shocks to introduce DNA into the cell.
• Cells are placed in suspension in an appropriate electroporation buffer
and put into an electroporation cuvette. DNA is added, the cuvette is
connected to a power supply, and the cells are subjected to a high-
voltage electrical pulse of defined magnitude and length. The cells are
then allowed to recover briefly before they are placed in normal cell
growth medium.

• Electroporation, also called electropermeabilization, is an efficient, non-viral

delivery system that allows genetic material (DNA and RNA), proteins, drugs or
other molecules to enter cells. It uses an accurately pulsed electrical current to
create temporary pores in the cell membrane through which the molecules
can then pass.
• Once inside, drugs and other molecules may act on the cell. Genetic material
may remain independent (as a plasmid) or become integrated into the host
genome depending on the downstream experimental steps.
 Liposome mediated
• Lipososme are artificial fluid filled spherical vesicles made of phospholipids
molecules.
• Produced from glycolipids, cholesterols, non toxic surfactants and
membranous proteins.
• These lipososmes work to deliver drug by diffusion rather than by direct cell
fusion.

• The walls of lipososme consist of continuous lipid bilayer organised in manner

as that of the bilayer of natural membrane.
• Drugs or DNA can be linked to the walls of the liposomes or contained at high
concentration within its lumen.
• Liposomes can protect from phagocytic cells of the immune system by
protective layer of synthetic polymer-polyethelene glycol.
• The walls of the liposome contains specific protein (hormones or antibodies)
that allow the liposome selectively to the surface of target cells.
• They can be preloaded with DNA by two common methods-membrane fusion
and endocytosis thus forming DNA-liposome complex.
• This complex fuses with the cell membrane of target cell and to release the
contents into the cell.
• Animal cell, plant cell, bacteria, yeast protoplasts are susceptible
to lipofection method.
 Liposomes can be classified
as either

Classification 1) Cationic liposome

2) Anionic liposome
Cationic liposome
• Positively charged liposome which associate with the negatively
charged DNA molecules by electrostatic interactions forming a
stable complex.
• The overall net positive charge allows the close association of the
lipoplex with the negatively charged cell membrane followed by
uptake into the cell and then into nucleus.
• Cationic lipids forming micellar structures called liposomes are
complexed with DNA to create lipoplexes.
• The structures fuse with the cell membrane, at least sometimes
after interactions with surface proteoglycans.
• The complexes are internalised by endocytosis, resulting in the
formation of a double-layer inverted micellar vesicle.
• During the maturation of the endosome into a lysosome, the
endosomal wall might rupture, releasing the contained DNA into
the cytoplasm and potentially towards the nucleus.
• DNA imported into the nucleus might result in gene expression.
Alternatively, DNA might be degraded within the lysosome.

Anionic liposome
• Some of the DNA molecules get entrapped within the aqueous
interior of these liposomes.
• Divalent cations are used (e.g. CA2+, Mg 2+, Mn2+ and Ba2+) which
can neutralize the mutual electrostatic repulsion.
• Cationic liposomes get inactivated and unstable in the presence of
serum and exhibit cytotoxicity.
• Due to reduced toxicity and interference from serum proteins, pH-
sensitive liposomes are considered as potential gene delivery
vehicles than the cationic liposomes.
 Microinjection
• Microinjection allows for the efficient transfer of controlled
nucleotide amounts into the nucleus of a specific target cell.
• It is a very precise, but time-consuming and expensive transfection
method with a very low throughput only.
• Microinjection is mostly used for special applications, such as single
cell manipulation or the generation of transgenic animals.

Principle:
• With this physical method, the target cell is positioned under a microscope and
being fixed by a pipette.
• The nucleotide solution is then directly injected into the cytoplasm and/or the
nucleus using a fine glass capillary needle.
• Once within the nucleus, the nucleotide can immediately integrate into the
endogenous DNA.
 Biolistic
• Biolistics, short for “biological ballistics” and also known as particle-mediated
gene transfer, is the method of directly shooting DNA fragments into cells
using a device called a gene gun.
• To use a gene gun, a scientist first mixes a DNA construct with particles of a
heavy metal, usually tungsten or gold. These fine particles stick to the
negatively charged DNA.
• The DNA/metal particles are loaded onto one side of a plastic bullet.
• A pressurized gas, usually helium, provides the force for the gun. Gas pressure
builds up until a rupture disk breaks, driving the plastic bullet down a shaft.
• The plastic bullet is abruptly stopped at the end of the shaft, but the
DNA/metal particles emerge from the gun with great speed and force.
• If the gun is aimed at biological tissue, some of the metal particles will
penetrate the cell membranes and deliver DNA constructs to cells.
 Vector mediated
method
• Vector-mediated gene
transfer is carried out
either by Agrobacterium-
mediated transformation
or by use of plant viruses
as vectors.

i. A. tumefaciens that
induces crown gall
There are mainly disease.
two species of
Agrobacterium:
ii. A. rhizogenes that
induces hairy root
disease.
A. tumefaciens that induces crown gall disease.

A. rhizogenes that induces hairy root disease.

 Methods of identification of recombinants:
• The DNA fragment was ligated in the vector of choice and
transformed into E. coli cells.
• The transformed cells were streaked across a culture plate resulting in
a multitude of colonies overnight.
• How do you know that all the steps have resulted in the correct
recombinant clones?
• Before you can declare the cloning victorious, colonies must be
screened for positive results.

Direct selection
Green fluorescent protein (GFP): which normally occurs in the
jellyfish Aequorea victoria, emits green light when exposed to ultraviolet light.
The gene for this protein is now widely used as a reporter gene
 Insertional inactivation
• Insertional inactivation is a technique
used in recombinant DNA technology.
• In this procedure, a bacteria carrying
recombinant plasmids or a fragment
of foreign DNA is made to insert into
a restriction site inside a gene to
resist antibiotics, hence causing the
gene to turn non-functional or in an
inactivated state.

Blue white selection:

• The β-galactosidase (lacZ) gene codes for an enzyme that can convert the
white substrate X-gal into a bright blue product.
• Many cloning plasmids contain the lacZ gene, along with genes for
antibiotic resistance.
• Foreign DNA can be inserted into the lacZ gene, inactivating it.
• Bacteria transformed with the plasmid are selected on a solid medium
containing X-gal and the appropriate antibiotic.
• Clones containing the recombinant plasmid cannot make β-
galactosidase and produce white colonies.
• Clones that contain the original plasmid with no insert express
the lacZ gene and make blue colonies.
Antibiotic resistance
• In this method , the selection of recombination relies on the presence of
an antibiotic resistance gene (ARG) in the plasmid vector.
• A marker gene helps in differentiating between transformant genes and
non-transformant genes.
• This helps in selecting the suitable recombinants.
• In case of E-coil; pBR322 is the vector for resistance to antibiotic
tetracycline.
• The insertional inactivation of pBR322 will result in loss of resistance to
tetracycline by E.coli.
• This can be found out by growing the recombinants on two plates; one
containing tetracycline and another containing ampicillin. The
recombinant will grow in ampicillin but not in tetracycline.
 Genomic library
What is a Genome?
• A genome is an organism’s complete set of DNA, including all of its genes. Each
genome contains all of the information needed to build and maintain that
organism. In humans, a copy of the entire genome is more than 3 billion DNA
base pairs which are present in all cell nuclei. The nuclear genome contains
protein-coding and non-coding genes, as well as other junk DNA and functional
regions of the genome.
• Most eukaryotes are diploid, which means that each chromosome has two
copies in the nucleus, but the term genome refers to just one copy of each
chromosome.
Genomic DNA Library
• A genomic library or gene bank is a complete collection of cloned DNA
fragments that constitutes the entire genome of an organism. It represents all
the genes – expressed, non-expressed, intron, exons, etc. Genomic libraries can
be kept for many years and the copies can be used for research purposes.
Steps to Construct a Genomic DNA Library
• First, the purification of the desired eukaryotic cell nuclei is done and this is
accomplished through protease digestion and organic extraction.
• The derived genomic DNA is too large to be incorporated into a vector and must be
fragmented into desired sizes. Both physical and enzymatic methods can be used to
fragment DNA.
• There are several vectors available for cloning large DNA fragments. Phage, bacterial
artificial chromosome, yeast artificial chromosome, and other such vectors are
suitable for larger DNA. The λ replacement vectors are the most preferred ones for
the construction of a genomic DNA library.
• Usually, the T4 DNA ligase enzyme is used to ligate the chosen DNA sequence into
the vector.
• The recombinant vectors and the insert combinations are grown in a bacterial host
cell (E. coli). They replicate their genome along with the vector genome contained
within them.
• The collection of clones that contain all the sequences from the original source
(including the sequence of interest) forms the gene bank or genomic library.

Screening of Clone
• Each transformed bacterial host cell in a library will
have only one vector with one DNA insert.
• The entire library can be plated over media on a filter.
• The filter, as well as colonies, are then hybridised,
labelled with a probe and identified using detection
methods such as autoradiography.
• Other techniques like PCR and the blue-white
selection method can also be used to screen the
clone.
cDNA library
• cDNA is created from a mature mRNA from a eukaryotic cell with the
use of reverse transcriptase.
• In eukaryotes, a poly-(A) tail (consisting of a long sequence of
adenine nucleotides) distinguishes mRNA from tRNA and rRNA and
can therefore be used as a primer site for reverse transcription.
• cDNA construction
• Once mRNA is purified, an oligo-dT primer (a short sequence of deoxy-
thymidine nucleotides) is bound to the poly-A tail of the RNA.
• The primer is required to initiate DNA synthesis by the enzyme reverse
transcriptase.
• This results in the creation of RNA-DNA hybrids.
• To remove the mRNA, the RNAse H enzyme is used to cleave the backbone
of the mRNA.
• DNA polymerase I is then added, the cleaved RNA acts as a primer the
DNA polymerase I can identify and initiate replacement of RNA nucleotides
with those of DNA.
• Restriction endonucleases and DNA ligase are then used to clone the
sequences into bacterial plasmids.
• The cloned bacteria are then selected, commonly through the use of
antibiotic selection. Once selected, stocks of the bacteria are created which
can later be grown and sequenced to compile the cDNA library.
cDNA Library uses:
• cDNA libraries are commonly used when reproducing eukaryotic
genomes, as the amount of information is reduced to remove the
large numbers of non-coding regions from the library.
• cDNA libraries are used to express eukaryotic genes in prokaryotes.
• Prokaryotes do not have introns in their DNA and therefore do not
possess any enzymes that can cut it out during transcription process.
cDNA does not have introns and therefore can be expressed in
prokaryotic cells.
• cDNA libraries are most useful in reverse genetics where the
additional genomic information is of less use.
• Additionally, cDNA libraries are frequently used in functional
cloning to identify genes based on the encoded protein's function.
cDNA Library vs. Genomic DNA Library
• cDNA library lacks the non-coding and regulatory elements found in
genomic DNA.
• cDNA libraries generally contain much smaller fragments than
genomic DNA libraries and are usually cloned into plasmid vectors.
• Genomic DNA libraries provide more detailed information about the
organism but are more resource-intensive to generate and keep.
• The genomic DNA libraries comprise large DNA fragments.

Construction of recombinant DNA molecule

Steps
• DNA of donor or gene of interest is isolated
• Cut using restriction endonucleases.
• The DNA of vector is cut into fragments using same RE.
• The two fragments are joined using DNA ligase.
• As a result Hybrid DNA or rDNA is obtained.
• rDNA is introduced into host cells such as E.coli, Bacillus subtilis,
Streptomyces sp.