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1. Recombinant DNA technology
Recombinant DNA Technology is defined as “the joining
together of DNA molecules from different organisms and
inserting it into a host organism to produce new genetic
combinations that are of value to science, medicine,
agriculture and industry.
Using this technology human insulin was produced initially
The first concept for recombinant DNA technology came
from Werner Arber’s.
From this discovery, geneticists learned to “cut” and
“paste” DNA molecules, and novel restriction enzymes for
cutting, and pasting were discovered or invented.

2. Recombinant DNA technology relates to the usage of three main
tools:
(1) Enzymes (restriction enzymes, polymerases, and ligases) - the
enzymes will help cut (restriction enzymes), synthesize (polymerases),
and bind (ligases) DNA. The restriction enzyme will cut at a specific site
within the DNA molecule called a restriction site and produce sticky
ends in the DNA sequence that will help it bind specifically to the
desired gene.
(2) Vectors - The vector will carry the desired gene. They are considered
as the final vehicles that carry genes of interest into the host organism,
the most commonly used vectors are plasmids and bacteriophages. A
vector must contain the same restriction sites that are found within the
desired gene to facilitate gene integration.
(3) Host organism. The host organism is the cell in which the
recombinant DNA is introduced and includes bacteria, fungi, and
animal cells. To introduce vectors into hosts, techniques involving
microinjection, biolistics, gene gun, alternate cooling and heating, and
calcium phosphate ions have been used.

3. In generally, a recombinant DNA technology has five steps:
•Selection and isolation of
DNA insert
•Selection of suitable
cloning vector
•Introduction of DNA-insert
into vector to form rec DNA
molecule
•Rec DNA molecule is
introduced into a suitable
host.
•Selection of transformed
host cells.
•Expression and
multiplication of DNA-
insert in the host.

4. (i) Selection and isolation of DNA insert: First step in rec DNA
technology is the selection of a DNA segment of interest which
is to be cloned. This desired DNA segment is then isolated
enzymatically. This DNA segment of interest is termed as DNA
insert or foreign DNA or target DNA or cloned DNA.
(ii) Selection of suitable cloning vector: A cloning vector is a self-
replicating DNA molecule, into which the DNA insert is to be
integrated. A suitable cloning vector is selected in the next step
of rec DNA technology. Most commonly used vectors are
plasmids and bacteriophages.
(iii) Introduction of DNA-insert into vector to form recDNA
molecule: The target DNA or the DNA insert which has been
extracted and cleaved enzymatically by the selective restriction
endonuclease enzymes [in step (i)] are now ligated (joined) by
the enzyme ligase to vector DNA to form a rec DNA molecule
which is often called as cloning-vector-insert DNA construct.

5. (iv) rec DNA molecule is introduced into a suitable host:
Suitable host cells are selected and the rec DNA molecule so
formed [in step (iii)] is introduced into these host cells. This
process of entry of rec DNA into the host cell is called
transformation. Usually selected hosts are bacterial cells like E.
coli, however yeast, fungi may also be utilized.
(v) Selection of transformed host cells: Transformed cells (or
recombinant cells) are those host cells which have taken up the rec
DNA molecule. In this step the transformed cells are separated
from the non-transformed cells by using various methods making
use of marker genes.
(vi) Expression and Multiplication of DNA insert in the host:
Finally, it is to be ensured that the foreign DNA inserted into the
vector DNA is expressing the desired character in the host cells.
Also, the transformed host cells are multiplied to obtain sufficient
number of copies. If needed, such genes may also be transferred
and expressed into another organism.

6. Tools for Recombinant DNA Technology:
•Enzymes:
1. Restriction Endonucleases,
2. Exonucleases,
3. DNA ligases,
•DNA polymerase
•Cloning Vector
•Host organism
•DNA insert or foreign DNA
•Linker and adaptor sequences.

7. 1. Restriction Endonuclease: These enzymes serve as
important tools to cut DNA molecules at specific sites,
which is the basic need for rec DNA technology. These are
the enzymes that produce internal cuts (cleavage) in the
strands of DNA, only within or near some specific sites
called recognition sites/recognition sequences/ restriction
sites 01 target sites. Such recognition sequences are
specific for each restriction enzyme.
Types of Restriction Endonucleases:
Type-I Restriction Endonucleases
Type-II Restriction Endonucleases
Type-III Restriction Endonucleases

8. Type-I Restriction Endonucleases: They require Mg++ ions and ATP for their
functioning. Such types of restriction endonucleases cleave the DNA about 1000 bp
away from the 5′ end of the sequence ‘TCA’ located within the recognition site.
Important examples of Type-I restriction endonuclease enzyme are EcoK, EcoB, etc.
Type-II Restriction Endonucleases:
These are most important endonucleases for gene cloning and hence for rec DNA
technology. These enzymes are most stable. They show cleavage only at specific sites
and therefore they produce the DNA fragments of a defined length. These enzymes
show cleavage in both the strands of DNA, immediately outs.de then- recognition
sequences. They require Mg++ions for their functioning.
Important examples of Type-II Restriction endonucleases include Hinfl, EcoRI, PvuII,
Alul, Haelll etc.
Type-III Restriction Endonucleases:
These are not used for gene cloning. They are the intermediate enzymes between Type-I
and Type-II restriction endonuclease. They require Mg++ ions and ATP for cleavage and
they cleave the DNA at well-defined sites in the immediate vicinity of recognition
sequences, e.g. Hinf III, etc.
Nature of cleavage by Restriction Endonucleases:
The nature of cleavage produced by a restriction endonuclease is of considerable
importance.

9. 2. Exonucleases:
o It removes nucleotides from the ends of a nucleic acid molecule. An
exonuclease removes nucleotide from the 5′ or 3′ end of a DNA
molecule. An exonuclease never produces internal cuts in DNA.
o Ex Bal31, E. coli exonuclease III, Lambda exonuclease, etc.
o Exonculease Bal31 are employed for making the DNA fragment with
blunt ends shorter from both its ends.
o E coli Exonuclease III is utilized for 3’end modifications because it
has the capability to remove nucleotides from 3′-OH end of DNA.
o Lambda exonuclease is used to modify 5′ ends of DNA as it removes
the nucleotides from 5′ terminus of a linear DNA molecule.
3. DNA ligase: The function of these enzymes is to join two fragments
of DNA by synthesizing the phosphodiester bond. They function to
repair the single stranded nicks in DNA double helix and in rec DNA
technology they are employed for sealing the nicks between adjacent
nucleotides. This enzyme is also termed as molecular glue.

10. DNA polymerases:
These are the enzymes which synthesize a new complementary DNA
strand of an existing DNA or RNA template. One such enzyme is DNA
polymerase 1 which , prepared from E coli and is employed to make the
protruding ends double-stranded by extension of the shorter strand.
Another type of DNA polymerase used in genetic engineering is Taq
DNA polymerase which is used in PCR (Polymerase Chain Reaction).
Reverse transcriptase is also an important type of DNA polymerase
enzyme for genetic engineering. It uses RNA as a template for
synthesizing a new DNA strand called as cDNA a e complementary
DNA). Its main use is in the formation of cDNA libraries. Apart from all
these above mentioned enzymes, a few other enzymes also mark their
importance in genetic engineering.

11. Cloning Vectors:
The cloning vector is the DNA molecule capable of replication in a host
organism, into which the target DNA is introduced producing the rec
DNA molecule.
A cloning vector may also be termed as a cloning vehicle or earner
DNA or simply as a vector or a vehicle a great variety of cloning vectors
are present for use with E. coli is the host organism.
Ex - bacteria like Bacillus, Pseudomonas, Agrobacterium, etc. and on
different eukaryotic organisms like yeast and other fungi.
The cloning vector which has only a single site for cutting by a particular
restriction endonuclease is Considered as a good cloning vector.
Different types of DNA molecules may be used as cloning vehicles such
as they may be plasmids, bacteriophages, cosmids, phasmids or artificial
chromosomes.

12. Host Organism: Most widely used host for rec DNA technology is the bacterium E.
coli. Because cloning and isolation of DNA inserts is very easy in this host.
oA good host organism is the one winch easy to transform and in which the replication
of rec DNA is easier.
oThere should not be any interfering element against the replication of rec DNA in the
host cells
DNA Insert Or Foreign DNA:
oThe desired DNA segment which is to be cloned is called as DNA insert or foreign
DNA or target DNA.
oThe selection of a suitable target DNA is the very first step of rec DNA technology.
oThe target DNA (gene) may be of viral, plant, animal or bacterial origin.
oFollowing points must be kept in mind while selecting the foreign DNA:
oCD It can be easily extracted from source.
oIt can be easily introduced into the vector.
oThe genes should be beneficial for commercial or research point of view.
oA number of foreign genes are being cloned for benefit of human beings. Some of
these DNA inserts are the genes responsible for the production of insulin, interferon’s,
lymphotoxins various growth factors, interleukins, etc.

13. Linker and Adaptor Sequences:
Linkers and adaptors are the DNA molecules which help in the modifications
of cut ends of DNA fragments. These can be joined to the cut ends and hence
produce modifications as desired.
Both are short, chemically synthesized, double stranded DNA sequences.
Linkers have (within them) one or more restriction endonuclease sites and
adaptors have one or both sticky ends.
Linkers contain target sites for the action of one or more restriction enzymes.
can be ligated to the blunt ends of foreign DNA or vector DNA.
Then they undergo a treatment with a specific restriction endonuclease to
produce cohesive ends of DNA fragments EcoRI-linker is a common example
of frequently used linkers.
Adaptors are the chemically Synthesized molecules which have pre-formed
cohesive ends.
Adaptors are employed for end modification in cases where the recognition
site for restriction endonuclease enzyme is present within the foreign DNA.
The foreign DNA is ligated with adaptor on both ends. This new molecule, so
formed, is then phosphorylated at the 5′-terminii. Finally foreign DNA modified
with adaptors is integrated into the vector DNA to form the recombinant DNA
molecule.

14. APPLICATIONS
Recombinant chymosin - Found in rennet, chymosin is an enzyme required to manufacture
cheese.
Recombinant human insulin
Recombinant human growth hormone(HGH, somatotropin) - Administered to patients
whose pituitary glands generate insufficient quantities to support normal growth and
development.
Recombinant blood clotting factor VIII – A blood-clotting protein that is administered to
patients with forms of the bleeding disorder hemophilia, who are unable to produce factor
VIII in quantities sufficient to support normal blood coagulation.
Recombinant hepatitis B vaccine - Hepatitis B infection is controlled through the use of a
recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface
antigen that is produced in yeast cells.
Diagnosis of infection with HIV - The antibody test (ELISA or western blot) uses a
recombinant HIV protein to test for the presence of antibodies that the body has produced in
response to an HIV infection.
Golden rice - A recombinant variety of rice that has been engineered to express the enzymes
responsible for β-carotene biosynthesis.
Herbicide-resistant crops - Commercial varieties of important agricultural crops (including
soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a
recombinant gene that results in resistance to the herbicide glyphosate(trade name Roundup),
and simplifies weed control by glyphosate application.
Insect-resistant crops - Bacillus thuringeiensis is a bacterium that naturally produces a
protein (Bt toxin) with insecticidal properties.