Recombinant DNA Technology and Genetically

Modified Organisms (GMOs)

What is Recombinant DNA Technology?

Recombinant DNA technology comprises altering genetic material outside an

organism to obtain enhanced desired characteristics in living organisms or their
products. This technology involves the insertion of DNA fragments from a variety of
sources, having a desirable gene sequence via an appropriate vector.

Fig. Recombinant DNA is made by combining DNA from

different sources.
Cutting of DNA molecule

Enzymes that recognize specific DNA sequences (usually 4 or 6 nucleotides) and

then cut the double-stranded DNA helix at this sequence. These enzymes are called
site-specific restriction endonucleases, or more simply “restriction enzymes”, and
they naturally function as part of bacterial defenses against viruses and other
sources of foreign DNA.

Style of DNA cutting by RE

The ends of a molecule cut by some of the restriction enzymes have an

overhanging region of single-stranded DNA, and so are sometimes called sticky
ends. On the other hand, some of them cut both strands in exactly the middle of the
recognition sequence, producing what are called blunt ends, which lack overhangs.
 Contd.

Fig. Example of how restriction enzymes cut DNA. (A) Treating the DNA with SmaI results
in fragments with blunt ends. (B) Whereas treatment with BamHI produces fragments with
“cohesive” or “sticky” ends.
Joining the DNA/Ligation of DNA

DNA Ligation is the joining of 2 DNA

molecules by the enzyme, DNA ligase.
DNA ligase catalyzes the formation of
two covalent phosphodiester bonds
between the 3’ hydroxyl group of one
nucleotide and the 5’ phosphate group of
another in an ATP-dependent reaction.
Molecular cloning
Molecular cloning is a set of experimental methods in molecular biology that are used
to assemble recombinant DNA molecules and to direct their replication within host
organisms.

Steps of cloning
In standard molecular cloning experiments, the cloning of any DNA fragment
essentially involves seven steps:
(1) Choice of host organism and cloning vector,
(2) Preparation of vector DNA,
(3) Preparation of DNA to be cloned,
(4) Creation of recombinant DNA,
(5) Introduction of recombinant DNA into the host organism,
(6) Screening for clones with desired DNA inserts and biological properties.
Choice of host organism and cloning vector
Although a very large number of host organisms and molecular cloning vectors are in
use, the great majority of molecular cloning experiments begin with a laboratory
strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E.
coli and plasmid vectors are in common use because they are
technically sophisticated, versatile, widely available, and offer rapid growth of
recombinant organisms with minimal equipment

If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of

base pairs), then a bacterial artificial chromosome or yeast artificial chromosome
vector is often chosen.
Preparation of vector DNA
The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site
where foreign DNA will be inserted. The restriction enzyme is chosen to generate a
configuration at the cleavage site that is compatible with the ends of the foreign DNA.
Typically, this is done by cleaving the vector DNA and foreign DNA with the same
restriction enzyme.
Preparation of DNA to be cloned
• DNA to be cloned is extracted from the organism of interest.
• Polymerase chain reaction (PCR) methods are often used for the amplification of
specific DNA
• cDNA cloning is usually used to obtain clones representative of the mRNA population
of the cells of interest,
• synthetic DNA is used to obtain any precise sequence defined by the designer. Such a
designed sequence may be required when moving genes across genetic codes (for
example, from the mitochondria to the nucleus) or simply for increasing expression via
codon optimization.
Creation of recombinant DNA with DNA ligase
The creation of recombinant DNA is in many ways the simplest step of the molecular
cloning process. DNA prepared from the vector and foreign source are simply mixed
together at appropriate concentrations and exposed to an enzyme (DNA ligase) that
covalently links the ends together. This joining reaction is often termed ligation. The
resulting DNA mixture containing randomly joined ends is then ready for introduction
into the host organism.

Introduction of recombinant DNA into host organism

The methods used to get DNA into cells are varied, and the name applied to this step in
the molecular cloning process will often depend upon the experimental method that is
chosen (e.g. transformation, transduction, transfection, electroporation).
Screening for clones with desired DNA inserts

This may be accomplished through a very wide range of experimental methods,

including-

• Alpha complementation test

• Insertional inactivation method
• Restriction digestion method
• Nucleic acid hybridizations
• Polymerase chain reaction
Genetically Modified Organism (GMO)
GMOs (genetically modified organisms) are organisms whose genetic fabric
(DNA) has been altered in a way that prevents it from occurring naturally through
mating or recombination. The word “genetic alteration” refers to the process of
altering an organism’s genetic makeup by introducing specific beneficial genes.
The genetic material of a genetically modified creature has been altered using
genetic engineering techniques.

The goal of Genetic modification

The goal of genetic modification is to provide enhanced features to plants by
altering their genetic makeup. This is done by inserting a novel gene or gene into
the genome of a plant. GMOs gain humanity when they are utilized for purposes
like improving the availability and quality of food and hospital therapy, as well as
contributing to a cleaner environment.
Creation of GMOs
Creating a genetically modified organism is a multi-step process. They
are:

• Identifying gene (s) of interest;

• Cloning the gene of interest (insertion of the gene(s) into vector and
transformation of vector);
• Selection of modified plant cells and their regeneration into full plants;
• Detection of the transformation and identification of the inserted DNA fragment:
• Performance testing of a plant;
• Conduct a risk assessment;
The benefits of GM organisms
Living organisms that have had their genes altered in some way are known as
GMOs. GMOs can be animals or microorganisms, but they’re most typically plants.
• Increase crop productivity;
• Advantages for the environment;
• GMOs are often low-cost;
• They increase the value of crops;
• They are known to lower the cost of meals;
• They produce products that have been found to be safe;
The negative effects of genetically modified organisms
• They would create plants that would linger in the soil for a long period of time,
leaving undesired lasting effects;
• They may represent a threat to insects that are beneficial to the environment;
• They have the potential to generate extra weeds and endanger the lives of
animals;
• They pose a hazard to crop range;
• They are suspected of doing shabby business in the agricultural industry;
• Antibiotic resistance may be exacerbated by GMOs;
• GMOs have also been linked to allergy responses;
Current Use of Genetically Modified Organisms
Agricultural plants are one of the most frequently cited examples of genetically
modified organisms (GMOs). Advances have also been made in developing crops
that mature faster and tolerate aluminum, boron, salt, drought, frost, and other
environmental stresses, allowing plants to grow in conditions where they might not
otherwise flourish.
Table: Examples of GMOs Resulting from Agricultural Biotechnology
Genetically Example Organism Genetic Change
Conferred

APPROVED COMMERCIAL PRODUCTS

Herbicide tolerance Soybean Glyphosate herbicide (Roundup) tolerance conferred by
expression of a glyphosate-tolerant form of the
plant enzyme 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS) isolated from the soil
bacterium Agrobacterium tumefaciens, strain CP4
Insect resistance Corn Resistance to insect pests, specifically the European corn
borer, through expression of the
insecticidal protein Cry1Ab from Bacillus thuringiensis
Altered fatty acid Canola High laurate levels achieved by inserting the gene for ACP
composition thioesterase from the California bay tree Umbellularia
californica
Virus resistance Plum Resistance to plum pox virus conferred by insertion of a coat
protein (CP) gene from the virus
 Contd.

Genetically Conferred Example Organism Genetic Change

APPROVED COMMERCIAL PRODUCTS

Vitamin enrichment Rice Three genes for the manufacture of beta-carotene, a
precursor to vitamin A, in the endosperm of the rice
prevent its removal (from husks) during milling
Vaccines Tobacco Hepatitis B virus surface antigen (HBsAg) produced in
transgenic tobacco induces immune response when
injected into mice
Oral vaccines Maize Fusion protein (F) from Newcastle disease virus (NDV)
expressed in corn seeds induces an immune response
when fed to chickens
Faster maturation Coho salmon A type 1 growth hormone gene injected into fertilized fish
eggs results in 6.2% retention of the vector at one year of
age, as well as significantly increased growth rates
Applications recombinant DNA technology

• Genome organization and gene expression

• Production of recombinant proteins
• Transgenic organisms
• Gene therapy
Genetically modified organisms

Bacteria: Bacteria were the first organisms to be genetically modified in the

laboratory, due to the relative ease of modifying their chromosomes. This ease made
them important tools for the creation of other GMOs. Genetically modified bacteria
are used to produce large amounts of proteins for industrial use. The majority of
these products are human proteins for use in medicine. The first medicinal use of GM
bacteria was to produce the protein insulin to treat diabetes. Other medicines
produced include clotting factors to treat hemophilia, human growth hormone to treat
various forms of dwarfism, interferon to treat some cancers, erythropoietin for
anemic patients, and tissue plasminogen activator which dissolves blood
clots. Outside of medicine, they have been used to produce biofuels.
Viruses

Most vaccines consist of viruses that have been attenuated, disabled, weakened, or
killed in some way so that their virulent properties are no longer effective. Genetic
engineering could theoretically be used to create viruses with virulent genes removed.
This does not affect the viruses’ infectivity, invokes a natural immune response and
there is no chance that they will regain their virulence function, which can occur with
some other vaccines. As such they are generally considered safer and more efficient
than conventional vaccines.
Outside of biology, scientists have used a genetically modified virus to construct
a lithium-ion battery and other nanostructured materials. They could be constructed at
lower temperatures with non-toxic chemicals, making them more environmentally
friendly.
Fungi

For industrial applications, yeasts combine the bacterial advantages of being a single-
celled organism that is easy to manipulate and grow with the advanced protein
modifications found in eukaryotes. They can be used to produce large complex
molecules for use in food, pharmaceuticals, hormones, and steroids. Yeast is important
for wine production and as of 2016 two genetically modified yeasts involved in the
fermentation of wine have been commercialized in the United States and Canada.
Button mushroom (Agaricus bisporus) has been gene edited to resist browning, giving it
a longer shelf life. The process used CRISPR to knock out a gene that
encodes polyphenol oxidase. As it didn't introduce any foreign DNA into the organism it
was not deemed to be regulated under existing GMO frameworks and as such is the first
CRISPR-edited organism to be approved for release.
Plants
Plants have been engineered for scientific research, to display new flower colors,
deliver vaccines, and create enhanced crops. Tobacco was the first plant to be altered
using genetic engineering and is considered a model organism for genetic
engineering. In research, plants are engineered to help discover the functions of certain
genes. The first genetically modified ornamentals commercialized altered
color. Carnations were released in 1997, with the most popular genetically modified
organism, a blue rose (actually lavender or mauve) created in 2004. The
papaya ringspot virus devastated papaya trees in Hawaii in the twentieth century until
transgenic papaya plants were given pathogen-derived resistance. A unique concern is
that a transgenic species may no longer bear enough resemblance to the original species
to truly claim that the original species is being conserved.
Crops:
Genetically modified crops are genetically modified plants that are used in agriculture.
The first crops developed were used for animal or human food and provided resistance
to certain pests, diseases, environmental conditions, spoilage or chemical treatments
(e.g. resistance to a herbicide). The second generation of crops aimed to improve the
quality, often by altering the nutrient profile. Third generation genetically modified
crops could be used for non-food purposes, including the production of pharmaceutical
agents, biofuels, and other industrially useful goods, as well as for bioremediation.

There are three main aims of agricultural advancement; increased production, improved
conditions for agricultural workers and sustainability. GM crops contribute by
improving harvests by reducing insect pressure, increasing nutrient value and tolerating
different abiotic stresses. Despite this potential, as of 2018, the commercialized crops
are limited mostly to cash crops like cotton, soybean, maize and canola and the vast
majority of the introduced traits provide either herbicide tolerance or insect resistance.
Animals
Mammals are the best models for human disease, making genetic engineered ones vital
to the discovery and development of cures and treatments for many serious diseases.
Knocking out genes responsible for human genetic disorders allows researchers to study
the mechanism of the disease and to test possible cures. Genetically modified mice have
been the most common mammals used in biomedical research, as they are cheap and
easy to manipulate.
Human proteins expressed in mammals are more likely to be similar to their natural
counterparts than those expressed in plants or microorganisms. The stable expression
has been accomplished in sheep, pigs, rats, and other animals. In 2009, the first human
biological drug produced from such an animal, a goat, was approved. The drug, ATryn,
is an anticoagulant that reduces the probability of blood clots during surgery or
childbirth and is extracted from goat's milk. Human alpha-1-antitrypsin is another
protein that has been produced from goats and is used in treating humans with this
deficiency. Livestock are modified with the intention of improving economically
important traits such as growth-rate, quality of meat, milk composition, disease
resistance and survival.
Animals have been engineered to grow faster, be healthier and resist
diseases. Modifications have also improved the wool production of sheep and the
udder health of cows. Goats have been genetically engineered to produce milk with
strong spiderweb-like silk proteins in their milk. Dairy cows have been genetically
engineered to produce milk that would be the same as human breast milk. This could
potentially benefit mothers who cannot produce breast milk but want their children to
have breast milk rather than formula. Researchers have also developed a genetically
engineered cow that produces allergy-free milk.
Humans

Gene therapy uses genetically modified viruses to deliver genes that can cure diseases
in humans. Although gene therapy is still relatively new, it has had some successes. It
has been used to treat genetic disorders such as severe combined
immunodeficiency, and Leber's congenital amaurosis. Treatments are also being
developed for a range of other currently incurable diseases, such as cystic
fibrosis, sickle cell anemia, Parkinson's disease, cancer, diabetes, heart
disease and muscular dystrophy. These treatments only affect somatic cells, meaning
any changes would not be inheritable. Germline gene therapy results in any change
being inheritable, which has raised concerns within the scientific community. In 2015,
CRISPR was used to edit the DNA of non-viable human embryos.
Fish
Genetically modified fish are used for scientific research, as pets and as a food
source. Through genetic engineering it is possible to increase growth rates, reduce food
intake, remove allergenic properties, increase cold tolerance and provide disease
resistance. Several groups have been developing zebrafish to detect pollution by
attaching fluorescent proteins to genes activated by the presence of pollutants. The fish
will then glow and can be used as environmental sensors. The GloFish is a brand of
genetically modified fluorescent zebrafish with bright red, green, and orange fluorescent
colors. It was originally developed by one of the groups to detect pollution, but is now
part of the ornamental fish trade, becoming the first genetically modified animal to
become publicly available as a pet when in 2003 it was introduced for sale in the USA.
Insects
In biological research, transgenic fruit flies (Drosophila melanogaster) are model
organisms used to study the effects of genetic changes on development. Fruit flies are
often preferred over other animals due to their short life cycle and low maintenance
requirements. Drosophila has been used to study genetics and inheritance, embryonic
development, learning, behavior, and aging. The discovery of transposons, in
particular the p-element, in Drosophila provided an early method to add transgenes to
their genome, although this has been taken over by more modern gene-editing
techniques.[265]
Due to their significance to human health, scientists are looking at ways to control
mosquitoes through genetic engineering. Malaria-resistant mosquitoes have been
developed in the laboratory by inserting a gene that reduces the development of the
malaria parasite.
Others
Systems have been developed to create transgenic organisms in a wide variety of other
animals. Chickens have been genetically modified for a variety of purposes. This
includes studying embryo development, preventing the transmission of bird flu etc.
Genetically modified frogs, in particular Xenopus laevis and Xenopus tropicalis, are
used in developmental biology research. GM frogs can also be used as pollution
sensors, especially for endocrine disrupting chemicals.
The nematode Caenorhabditis elegans is one of the major model organisms for
researching molecular biology. RNA interference (RNAi) was discovered in C.
elegans and could be induced by simply feeding them bacteria modified to
express double-stranded RNA.
Transformation: When microorganisms are able to take up and replicate DNA from
their local environment, the process is termed transformation, and cells that are in a
physiological state such that they can take up DNA are said to be competent.

Transfection: The process of introducing DNA into cells is commonly termed

transfection.

Electroporation: Electroporation uses high-voltage electrical pulses to translocate

DNA across the cell membrane (and cell wall, if present).

Transduction: transduction involves the packaging of DNA into virus-derived

particles, and using these virus-like particles to introduce the encapsulated DNA into
the cell through a process resembling viral infection.