2.  Deoxyribonucleic acid (DNA) is an informational
molecule encoding the genetic instructions used in the
development and functioning of all known
living organisms and many viruses.
 Recombinant DNA (rDNA) molecules are DNA sequences
that result from the use of laboratory methods (molecular
cloning) to bring together genetic material from multiple
sources, creating sequences that would not otherwise be
found in biological organisms.
 Recombinant DNA technology is the technology of
preparing recombinant DNA in vitro by cutting up DNA
molecules and splicing together fragments from more than
one organism.

3. How is Recombinant DNA made?
There are three different methods by which
Recombinant DNA is made.
They are -
1. Transformation
2. Phage Introduction
3. Non-Bacterial Transformation.

4. Transformation
 The first step in transformation is to select a piece of
DNA to be inserted into a vector.
 The second step is to cut that piece of DNA with a
restriction enzyme and then ligate the DNA insert into
the vector with DNA Ligase.
 The insert contains a selectable marker which allows
for identification of recombinant molecules.
 The vector is inserted into a host cell, in a process
called transformation. One example of a possible host
cell is E. Coli. The host cells must be specially
prepared to take up the foreign DNA.

5. Non-bacterial Transformation
 This is a process very similar to Transformation,
The only difference between the two is non-bacterial
does not use bacteria such as E. Coli for the host.
 In microinjection, the DNA is injected directly into the
nucleus of the cell being transformed.
 In biolistics, the host cells are bombarded with high
velocity microprojectiles, such as particles of gold or
tungsten that have been coated with DNA.

6. Phage Introduction
 Phage introduction is the process of transfection, which is
equivalent to transformation, except a phage is used
instead of bacteria. In vitro packagings of a vector is
used. This uses lambda or MI3 phages to produce phage
plaques which contain recombinants. The recombinants
that are created can be identified by differences in
the recombinants and non-recombinants using various
selection methods.

7. Why is rDNA important?
Recombinant DNA has been gaining in importance over the last few
years, and recombinant DNA will only become more important in the
21st century as genetic diseases become more prevalent and
agricultural area is reduced. Below are some of the areas where
Recombinant DNA will have an impact -
 Better Crops (drought & heat resistance)
 Recombinant Vaccines (i.e. Hepatitis B)
 Prevention and cure of sickle cell anemia
 Prevention and cure of cystic fibrosis
 Production of clotting factors
 Production of insulin
 Production of recombinant pharmaceuticals
 Plants that produce their own insecticides
 Germ line and somatic gene therapy

8. Advantages of Recombinant DNA
Technology
Benefits include engineering organisms that have a desirable trait. For example,
insulin for many decades was harvested from animal pancreases.
Recombinant bacteria have been designed that produce human insulin in large
quantities, so animal pancreases are no longer required. Gene therapy is
another benefit, as it seeks to supplement mutant copies of genes with the correct
copies. A success in gene therapy is the recent treatment of metastatic
melanoma (cancer) with gene-therapy. Genetically modified crops with traits
such as resistance to disease and insects or other desirable characteristics.
 Provide substantial quantity
 No need for natural or organic factors
 Tailor made product that you can control
 Unlimited utilizations
 Cheap
 Resistant to natural inhibitors

9. Disadvantages of Recombinant DNA
Technology
A drawback is that modified organisms can spread through
nature and interbreed with natural organisms, thereby
contaminating environments and future generations in an
unforeseeable and uncontrollable way. Also, the interactions of
the designer genes with the wild-type genes can be unpredictable.
 Commercialized and became big source of income for
businessmen
 Effects natural immune system of the body
 Can destroy natural ecosystem that relies on organic cycle
 Prone to cause mutation that could have harmful effects
 Major international concern: manufacturing of biological
weapons such as botulism & anthrax to target humans with
specific genotype
 Concerns of creating super‐human race

10. Production of Insulin
The first step in creating synthetic human insulin is to extract human DNA. The
method to extracting human DNA is as follows:
 First charge a sample of blood, normally between 300 and 2500 microlitres, and allow it
to undergo a treatment of lysis by a cationic detergent, for example
tetradecyltrimethilammoniumbromide (TTAB) or dodecyltrimethilammoniumbromide
(DTAB) both with sodium chloride at a concentration higher than 0.5 M. Then mix up
the solution and heat it up to 68 C and incubate it for five minutes. Add 1 volume of
chloroform or another organic solvent. Allow the mixture to undergo centrifuging with a
normal bench centrifuge for a few minutes to eliminate the protein portion which forms
a clog with the organic base.
 After centrifuging, to the aqueous phase add a quantity of water to decrease the ionic
strength below 0.5 M NaCl, and a cationic detergent (for instance a solution of 5% of
Cetyl-trimetil-ammonium bromide), so that precipitation of the cationic detergent
micellar complex-DNA takes place after a short mixing operation. The solution now
containing the micellar-DNA complex then undergoes filtration. The ultra filtration takes
place with a filter (for instance, sintered borosilicate glass or an organic matrix like
polypropilene or polyethylene) of known and tested porosity (pore size between 5 and
15 micron), which retains the DNA-cationic detergent micellar complex in a satisfactory
way. The hydrophilic surface enables DNA to be recovered easily and speedily after the
washing operations. The organic matrix filter allows a slower recovery of DNA so it is
not commonly used at the moment. As genomic DNA is immobilized on the filter, it is
then eluted. (Schneider, 1995).

12.  Once the human DNA has been extracted, it is necessary to isolate the exact gene for
insulin. It is located in the top of the short arm of the eleventh chromosome. The DNA
sequence for the A chain is compromised of sixty three nucleotides and the sequence for
the B chain is compromised of ninety nucleotides. An extra codon must be placed at the
end of both sequences to signal the termination of protein synthesis. Also, an anti-codon,
consisting of the amino acid methionine, must be placed at the beginning of each chain in
order to make the removal of the insulin possible. (Singh-Khaira, 1994).
 In order to “cut” the genes for insulin production from the DNA you must mix the human
DNA with the enzymes HincII and BamHI (Laub, Rall, Bell, Rutter, 1982).
 Once the DNA has been cleaved and the insulin gene and been isolated, the mixture must
be run through an agarose gel electrophoresis in order to be able to remove the insulin
gene. First you must cast the agarose gel. To do this place the well-forming comb into the
gel-casting tray and pour enough agarose solution to fill the tray to 6mm.
 After the gel has set (15-20 minutes), place it into the gel box, fill the box with enough
TBE buffer to cover the gel, and remove the comb. After these steps have been completed,
the DNA must be loaded into the gel by inserting it into the wells with a pipet. After all of
the DNA has been loaded, close the electrophoresis chamber and connect the electrical
leads to a power supply and turn it on. Wait until the bands are almost to the end and then
remove the gel and stain it (Owen, 2000). Once the gel has been stained, elute the DNA
fragments by shaking the gel in a solution of 0.2 N NaCl, 1mm EDTA, and 10 mm Trk
(Laub, 1982).

14.  Now that the insulin gene has been isolated and removed, it
must be inserted into the plasmid of the vector cell, which is
E. coli. The genes for the A and B chains of insulin are
inserted into the gene for the bacterial enzyme, B-
galactosidase. Once the genes have been inserted into the
plasmid, the plasmid is reinserted into the E. coli cell.
 When the cell replicates, the B-galactosidase is formed with
either the A or B chain of insulin attached to it. The two
chains are then extracted and purified. Then they are mixed
together thus connecting to each other by forming disulfide
cross bridges. The end result is Humulin, or synthetic human
insulin (Singh-Khaira, 1994).

16. Scopes
 Agriculture: Growing crops of your choice (GM food),
pesticide resistant crops, fruits with attractive colors,
all being grown in artificial conditions.
 Pharmacology: Artificial insulin production, drug
delivery to target sites.
 Medicine: Gene therapy, antiviral therapy, vaccination,
synthesizing clotting factors.
 Other uses: Fluorescent fishes, glowing plants etc.

17. Environmental Conversation
 The use of recombinant DNA technology to control feral pests
such as the European rabbit (Oryctolagus cuniculus) is
undergoing research.
 Rabbits have developed a resistance to the myxomatosis virus.
A naturally occurring rabbit gene (ZPC ) affects the zona
pellucida and blocks fertilization in rabbits. This gene has been
isolated and added to the myxomatosis virus. The resulting
recombinant virus, acting as a vector for ZPC, has been very
successful under laboratory conditions as a contraceptive. If
released into the wild it may reduce the rabbit population and
the rabbit's status as a major pest.
 Another example of environmental clean-up and conservation
using recombinant DNA technology is the use of bacteria in
clearing areas containing landmines.

18. Use in Agriculture
 One of the most important applications of
biotechnology is in the production of food. Crop plants
and livestock have been genetically modified using
recombinant DNA techniques to increase yield,
improve quality and develop new varieties that are
resistant to disease, or can survive in arid or high salt
conditions.

19. Recombinant Vaccines
 A viral disease called infectious bursal disease (IBD) attacks the
white blood cells that normally produce antibodies in chicken.
This weakens the immune system and makes chickens
susceptible to other diseases.
 A protein from the virus causing IBD was found to produce a
high immune response, i.e. the production of antibodies. The
gene that produces the viral protein has been isolated and
introduced into a bacterial plasmid. The plasmid, now
containing recombinant DNA, is inserted into a bacterial cell
and cloned. Large quantities of the viral protein are produced
and form the basis of a vaccine for IBD in chickens.
 Protein-based vaccines for dealing with footrot in sheep and
cattle tick in cattle have also been developed in this way.

20. Use in Medicine
 Human enzymes, like insulin or human growth hormone, are created
in normally functioning bodies. Enzymes are proteins and proteins
are made up of a specific sequence of amino acids. The amino acid
sequence is determined by the person's DNA. Previously, diabetics
used insulin from pigs but it was not accepted well by all diabetics as
the amino acid sequence was slightly different. Now, scientists have
developed bacteria which have had the human gene for insulin
inserted into them using recombinant DNA techniques. Since the
amino acid sequence is the same, diabetics readily accept it even
though it was manufactured by bacteria. In a similar
fashion, scientists have developed protocols for the production of
clotting factors, hGH, virus fighting proteins, and many other
medicines are in development.
 Genetic engineering has resulted in a series of medical products. The
first two commercially prepared products from recombinant DNA
technology were insulin and human growth hormone, both of which
were cultured in the E. coli bacteria.

21. Use in Bioplastics
Regular plastics are a polymer created from
petroleum, which is a nonrenewable fossil fuel. When we
run out of petroleum, we could run out of plastic, not just
gas for our cars. This possibility has spurred the science of
creating bioplastics. Bioplastics are created from products
produced by plants, often through means of recombinant
DNA. Scientists have engineered a gene that will produce
a compound nearly identical to commercial plastics and
it's application is a hot area of research today. If
successful, scientists will have ensured that we will never
run out of plastic or have to worry about the pollution of
creating plastic from old, nonrenewable technologies.

22. Use in Pharmaceutical Products
 By recombinant DNA technology discussed previously, human genes
for various proteins have been engineered into expression vectors
and then into bacterial host cells that produce and secrete the protein.
Examples include insulin, used to treat diabetes, and human growth
hormone, used to treat hypopituitarism, which causes a form of
dwarfism.
 Another example is tissue plasminogen activator (TPA), which helps
dissolve blood clots and reduces the risk of subsequent heart attacks
if administered shortly after an initial attack. It is also possible to
construct desired molecules.
 Genetically engineered proteins can block or mimic surface receptors
on cell membranes; an example is a molecule designed to mimic a
receptor protein that HIV binds to in entering white blood cells (if
HIV binds to the drug molecules instead of those on the cell surface,
it would fail to enter the blood cell).
 Recombinant DNA techniques can generate large amounts of
proteins associated with the immune response against pathogens or
be used to modify the genome of a pathogen to attenuate it, and thus
lead to more specific and safer vaccines.