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GENETIC ENGINEERING
FEBRUARY 7, 2013
MRS. HAUGHTON
CAPE BIOLOGY
• Genetic engineering,
also known
as recombinant DNA
technology, means
altering the genes in a
living organism to
produce a Genetically
Modified Organism
(GMO) with a new
genotype.
•
1.
2.
3.
4.
Various kinds of genetic modification are
possible:
inserting a foreign gene from one
species into another
forming a transgenic organism
altering an existing gene so that its
product is changed
changing gene expression so that it is
translated more often or not at all.
TECHNIQUES OF GENETIC
ENGINEERING
• Genetic engineering is a very young
discipline, and is only possible due to the
development of techniques from the 1960s
onwards.
• These techniques have been made
possible from our greater understanding of
DNA and how it functions following the
discovery of its structure by Watson and
Crick in 1953.
• Although the final goal of genetic
engineering is usually the
expression of a gene in a host, in
fact most of the techniques and
time in genetic engineering are
spent isolating a gene and then
cloning it.
TECHNIQUE
PURPOSE
Restriction
enzymes
To cut DNA at specific points,
making small fragments
DNA ligase
To join DNA fragments together
Vectors
To carry DNA into cells and
ensure replication
Plasmids
Common kind of vector
Genetic markers To identify cells that have been
transformed
TECHNIQUE
PURPOSE
Polymerase chain
reaction (PCR)
To amplify very small pieces of DNA
cDNA
To make a DNA copy of RNA
DNA probes
To identify and label a piece of DNA
containing a certain sequence
Gene synthesis
To make a gene from scratch
Electrophoresis
To separate fragments of DNA
DNA Sequencing
To read the base sequence of a
length of DNA
RECOMBINANT DNA
TECHNOLOGY
• Genetic engineering in bacteria can be
broken down into six stages:
1
• Recombinant technology begins with
the isolation of a gene of interest. The
gene is then inserted into a vector
and cloned.
2
• A vector is a piece of DNA that is capable
of independent growth; commonly used
vectors are bacterial plasmids and viral
phages.
• The gene of interest (foreign DNA) is
integrated into the plasmid or phage, and
this is referred to as recombinant DNA.
3
• Before introducing the vector containing
the foreign DNA into host cells to express
the protein, it must be cloned. Cloning is
necessary to produce numerous copies of
the DNA since the initial supply is
inadequate to insert into host cells.
5
• Once the vector is isolated in large
quantities, it can be introduced into the
desired host cells such as mammalian,
yeast, or special bacterial cells.
• The host cells will then synthesize the
foreign protein from the recombinant DNA.
6
• When the cells are grown in vast
quantities, the foreign or recombinant
protein can be isolated and purified in
large amounts.
Other uses for recombinant DNA
• Recombinant DNA technology is not only
an important tool in scientific research, but
it has also impacted the diagnosis and
treatment of diseases and genetic
disorders in many areas of medicine.
• It has enabled many advances, including:
Isolation of large quantities
of protein
• In addition to the folliclestimulating hormone, insulin,
growth hormone and other
proteins are now available as
recombinant products.
Identification of mutations
• People may be tested for the
presence of mutated proteins that
may be associated with breast
cancer, retino-blastoma, and
neurofibromatosis.
Diagnosis of affected and carrier
states for hereditary diseases
• Tests exist to determine if people are
carriers of the cystic fibrosis gene, the
Huntington’s disease gene, the Tay-Sachs
disease gene, or the Duchenne muscular
dystrophy gene.
Transferring of genes from one
organism to another
• People suffering from cystic fibrosis,
rheumatoid arthritis, vascular disease, and
certain cancers may now benefit from the
progress made in gene therapy.
Mapping of human genes on
chromosomes
• Scientists are able to link mutations and
disease states to specific sites on
chromosomes.
RESTRICTION
ENDONUCLEASES
• Restriction enzymes are DNA-cutting
enzymes found in bacteria (and harvested
from them for use).
• Because they cut within the molecule, they
are often called restriction
endonucleases.
• In order to be able to sequence DNA, it is first
necessary to cut it into smaller fragments.
• Many DNA-digesting enzymes (like those in your
pancreatic fluid) can do this, but most of them
are not used for sequence work because they
cut each molecule randomly.
• This produces a heterogeneous collection of
fragments of varying sizes.
• What is needed is a way to cleave the DNA
molecule at a few precisely-located sites so that
a small set of homogeneous fragments are
produced.
• The tools for this are the restriction
endonucleases. The rarer the site it recognizes,
the smaller the number of pieces produced by a
given restriction endonuclease.
• A restriction enzyme recognizes and cuts DNA
only at a particular sequence of nucleotides.
• For example, the bacterium Hemophilus
aegypticus produces an enzyme
named HaeIII that cuts DNA wherever it
encounters the sequence 5'GGCC3'
3'CCGG5‘.
• The cut is made between the adjacent G and C.
• This particular sequence occurs at 11 places in
the circular DNA molecule of the virus φX174.
• Thus treatment of this DNA with the enzyme
produces 11 fragments, each with a precise
length and nucleotide sequence.
• These fragments can be separated from one
another and the sequence of each determined.
• HaeIII and AluI cut straight across the double
helix producing "blunt" ends. However, many
restriction enzymes cut in an offset fashion.
• The ends of the cut have an overhanging piece
of single-stranded DNA. These are
called "sticky ends" because they are able to
form base pairs with any DNA molecule that
contains the complementary sticky end.
• Any other source of DNA treated with the same
enzyme will produce such molecules.
• Mixed together, these molecules can join
with each other by the base pairing
between their sticky ends. The union can
be made permanent by another enzyme,
DNA ligase, that forms covalent bonds
along the backbone of each strand.
• The result is a molecule of recombinant
DNA (rDNA).
• Recombinant DNA molecules have
revolutionized the study of genetics and laid the
foundation for much of the biotechnology
industry.
• The availability of human insulin (for diabetics),
human factor VIII (for males with hemophilia A),
and other proteins used in human hormone
therapy all were made possible by recombinant
DNA.
• Restriction enzyme 1
• http://www.youtube.com/watch?v=aA5fyW
Jh5S0
• http://www.youtube.com/watch?v=-sI5vycD2g
Applications of Recombinant
DNA Technology
Human Applications
• Treatment of genetic disorders. Medical
scientists now know of about 3,000 disorders
that arise because of errors in an individual's
DNA.
• Conditions such as sickle-cell anemia, TaySachs disease, Duchenne muscular dystrophy,
Huntington's chorea, cystic fibrosis, and LeschNyhan syndrome are the result of the loss,
mistaken insertion, or change of a single
nitrogen base in a DNA molecule.
•
• Genetic engineering makes it possible for scientists to
provide individuals who lack a certain gene with correct
copies of that gene.
• For instance, in 1990 a girl with a disease caused by a
defect in a single gene was treated in the following
fashion. Some of her blood was taken, and the missing
gene was copied and inserted into her own white blood
cells, then the blood was returned to her body.
• If—and when—that correct gene begins to function, the
genetic disorder may be cured. This type of procedure is
known as human gene therapy (HGT)
Agricultural Applications
• It is now possible to produce plants that
will survive freezing temperatures, take
longer to ripen, convert atmospheric
nitrogen to a form they can use,
manufacture their own resistance to pests,
and so on.
• By 1988 scientists had tested more than
two dozen kinds of plants engineered to
have special properties such as these.
• Domestic animals have been genetically
"engineered" in an inexact way through
breeding programs to create more meaty
animals, etc., but with genetic engineering,
these desirable traits could be guaranteed
for each new generation of animal.
GENE THERAPY
• Gene therapy is the insertion, alteration,
or removal of genes within an
individual's cells and biological tissues to
treat disease.
• It is a technique for correcting defective
genes that are responsible for disease
development.
• The most common form of gene therapy
involves the insertion of functional genes into an
unspecified genomic location in order to replace
a mutated gene, but other forms involve directly
correcting the mutation or modifying normal
gene that enables a viral infection.
• Although the technology is still in its infancy, it
has been used with some success.
Types of gene therapy
• Gene therapy may be classified into the
two following types:
• Germ line gene therapy
• Somatic gene therapy
Germ line gene therapy
• In the case of germ line gene therapy,
germ cells, i.e., sperm or eggs, are
modified by the introduction of functional
genes, which are integrated into their
genomes.
• Therefore, the change due to therapy
would be heritable and would be passed
on to later generations.
Germ line gene therapy
• This new approach, theoretically, should
be highly effective in counteracting genetic
disorders and hereditary diseases.
However, many jurisdictions prohibit this
for application in human beings, at least
for the present, for a variety of technical
and ethical reasons.
Somatic gene therapy
• In the case of somatic gene therapy, the
therapeutic genes are transferred into
the somatic cells of a patient.
• Any modifications and effects will be
restricted to the individual patient only, and
will not be inherited by the patient's
offspring or later generations.
ETHICS
DESIGNER BABIES
Vectors in gene therapy
Viruses
• All viruses bind to their hosts and
introduce their genetic material into the
host cell as part of their replication cycle.
• This genetic material contains basic
'instructions' of how to produce more
copies of these viruses, hijacking the
body's normal production machinery to
serve the needs of the virus.
Retroviruses
• The genetic material in retroviruses is in
the form of RNA molecules, while the
genetic material of their hosts is in the
form of DNA.
• When a retrovirus infects a host cell, it will
introduce its RNA together with some
enzymes, namely reverse transcriptase
and integrase, into the cell.
Retroviruses
• This RNA molecule from the retrovirus
must produce a DNA copy from its RNA
molecule before it can be integrated into
the genetic material of the host cell.
Adenoviruses
• Adenoviruses are viruses that carry their genetic
material in the form of double-stranded DNA.
• They cause respiratory, intestinal, and eye
infections in humans (especially the common
cold).
• When these viruses infect a host cell, they
introduce their DNA molecule into the host.
• The genetic material of the adenoviruses is not
incorporated (transient) into the host cell's
genetic material.
Adenoviruses
• The DNA molecule is left free in the nucleus of
the host cell, and the instructions in this extra
DNA molecule are transcribed just like any other
gene.
• The only difference is that these extra genes are
not replicated when the cell is about to undergo
cell division so the descendants of that cell will
not have the extra gene.
• As a result, treatment with the adenovirus will
require re-administration in a growing cell
population.
Injection of Naked DNA
• This is the simplest method of non-viral
transfection.
• Cellular uptake of naked DNA is generally
inefficient.
• Research efforts focusing on improving the
efficiency of naked DNA uptake have yielded
several novel methods, such as the use of
a "gene gun", which shoots DNA coated gold
particles into the cell using high pressure gas.
Physical Methods to
Enhance Delivery
Electroporation
• Electorporation is a method that uses
short pulses of high voltage to carry DNA
across the cell membrane.
• This shock is thought to cause temporary
formation of pores in the cell membrane,
allowing DNA molecules to pass through.
• Electroporation is generally efficient and
works across a broad range of cell types.
However, a high rate of cell death
following electroporation has limited its
use, including clinical applications.
Gene Gun
• The use of particle bombardment, or
the gene gun, is another physical method
of DNA transfection.
• In this technique, DNA is coated with gold
particles and loaded into a device which
generates a force to achieve penetration
of DNA/gold into the cells.
Sonoporation
• Sonoporation uses ultrasonic frequencies
to deliver DNA into cells. The process of
acoustic cavitation is thought to disrupt the
cell membrane and allow DNA to move
into cells.
Magnetofection
• In a method termed magnetofection, DNA
is complexed to a magnetic particles, and
a magnet is placed underneath the tissue
culture dish to bring DNA complexes into
contact with a cell monolayer.
Problems and ethics
• Short-lived nature of gene therapy – Before
gene therapy can become a permanent cure for
any condition, the therapeutic DNA introduced
into target cells must remain functional and the
cells containing the therapeutic DNA must be
long-lived and stable.
• Immune response – Anytime a foreign object is
introduced into human tissues, the immune
system has evolved to attack the invader.
• Problems with viral vectors – Viruses, the
carrier of choice in most gene therapy
studies, present a variety of potential
problems to the patient —toxicity, immune
and inflammatory responses, and gene
control and targeting issues. In addition,
there is always the fear that the viral
vector, once inside the patient, may
recover its ability to cause disease.
• Multigene disorders – Conditions or disorders
that arise from mutations in a single gene are
the best candidates for gene therapy.
• Chance of inducing a tumor (insertional
mutagenesis) - If the DNA is integrated in the
wrong place in the genome, for example in
a tumor suppressor gene, it could induce a
tumor.
• Deaths may occur.
• What are the ethical issues surrounding gene
therapy?
• Because gene therapy involves making changes to the
body’s set of basic instructions, it raises many unique
ethical concerns. The ethical questions surrounding
gene therapy include:
• How can “good” and “bad” uses of gene therapy be
distinguished?
• Who decides which traits are normal and which
constitute a disability or disorder?
• Will the high costs of gene therapy make it available only
to the wealthy?
• Could the widespread use of gene therapy make society
less accepting of people who are different?
• Should people be allowed to use gene therapy to
enhance basic human traits such as height, intelligence,
or athletic ability?
PROS OF GENETIC
ENGINEERING
• Crops like potato, tomato, soybean and rice are
currently being genetically engineered to obtain new
strains with better nutritional qualities and increased
yield.
• The genetically engineered crops are expected to
have a capacity to grow on lands that are presently
not suitable for cultivation.
• The manipulation of the genes in crops is expected
to improve their nutritional value as also their rate of
growth. Biotechnology, the science of genetically
engineering foods, can be used to impart a better
taste to certain foods.
• Engineered seeds are resistant to pests and can
survive in a relatively harsh climatic conditions.
• The recently identified plant gene known as AtDBF2, when inserted in tomato and tobacco
cells is seen to increase their endurance to
harsh soil and climatic conditions.
• Biotechnology can be used to slow down the
process of food spoilage. It can thus result in
fruits and vegetables having a greater shelf life.
• Genetic engineering in food can be used
to produce totally new substances such as
proteins and other food nutrients. The
genetic modification of foods can be used
to increase their medicinal value, thus
making available homegrown edible
vaccines.
• Genetic engineering has a great potential
of succeeding in case of human beings.
This specialized branch of genetic
engineering, which is known as human
genetic engineering is the science of
modifying the genotypes of human beings
before birth. The process can be used to
manipulate certain traits in an individual.
• Positive genetic engineering deals with
enhancing the positive traits in an individual like
increasing longevity or human capacity while
negative genetic engineering deals with the
suppression of the negative traits in human
beings like certain genetic diseases. Genetic
engineering can be used to obtain a permanent
cure for certain dreaded diseases.
• If the genes responsible for the
exceptional qualities in some individuals
can be discovered, these genes can be
artificially introduced into genotypes of
other human beings. Genetic engineering
in human beings can be used to change
the DNA of individuals to bring about
desirable structural and functional
changes in them.
CONS OF GENETIC
ENGINEERING
• Genetic engineering in food involves the
contamination of genes in crops.
Genetically engineered crops may
supersede the natural weeds; they may
prove harmful for the natural plants.
Undesirable genetic mutations can lead to
allergies in crops. Critics believe that
genetic engineering in foodstuffs can
rather hamper the nutritional value while
enhancing their taste and appearance.
• Horizontal gene transfer can give rise to
new pathogens. While increasing the
immunity to diseases in plants, the
resistance genes may get transferred to
the harmful pathogens.
• Gene therapy in human beings can
manifest certain side effects. While
treating one defect, the therapy may lead
to another. As one cell is responsible for
many characteristics, the isolation of the
cells responsible for a single trait is indeed
difficult.
• Genetic engineering can hamper the
diversity in human beings. Cloning can be
detrimental to individuality. Moreover, such
processes may not be affordable for the
masses, thus making gene therapy, an
impossibility for the common man.
• Genetic engineering may work wonders
but it is after all a process of manipulating
the nature. It is altering something that is
not an original human creation. Modifying
something that one has not created is
always challenging.