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Title: GENETIC ENGINEERING


1
GENETIC ENGINEERING
  • FEBRUARY 7, 2013
  • MRS. HAUGHTON
  • CAPE BIOLOGY

2
  • 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.

3
  • 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. 

4
TECHNIQUES OF GENETIC ENGINEERING
5
  • 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.

6
  • 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.

7
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
8
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
9
RECOMBINANT DNA TECHNOLOGY
10
  • Genetic engineering in bacteria can be broken
    down into six stages

11
1
  • Recombinant technology begins with the isolation
    of a gene of interest. The gene is then inserted
    into a vector and cloned.

12
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.

13
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.

14
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.

15
6
  • When the cells are grown in vast quantities, the
    foreign or recombinant protein can be isolated
    and purified in large amounts.

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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

19
Isolation of large quantities of protein
  • In addition to the follicle-stimulating hormone,
    insulin, growth hormone and other proteins are
    now available as recombinant products. 

20
Identification of mutations
  • People may be tested for the presence of mutated
    proteins that may be associated with breast
    cancer, retino-blastoma, and neurofibromatosis. 

21
Diagnosis of affected and carrier states for
hereditary diseases
  • Tests exist to determine if people are carriers
    of the cystic fibrosis gene, the Huntingtons
    disease gene, the Tay-Sachs disease gene, or the
    Duchenne muscular dystrophy gene. 

22
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. 

23
Mapping of human genes on chromosomes
  • Scientists are able to link mutations and disease
    states to specific sites on chromosomes.

24
RESTRICTION ENDONUCLEASES
25
  • 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.

26
  • 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.

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  • 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.

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  • 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.

31
  • This particular sequence occurs at 11 places in
    the circular DNA molecule of the virus fX174.
  • 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.

32
  • 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.

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  • 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).

35
  • 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.

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  • Restriction enzyme 1
  • http//www.youtube.com/watch?vaA5fyWJh5S0
  • http//www.youtube.com/watch?v-sI5vy-cD2g
  • Restriction enzyme 1
  • http//www.youtube.com/watch?vaA5fyWJh5S0
  • http//www.youtube.com/watch?v-sI5vy-cD2g

38
Applications of Recombinant DNA Technology
39
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, Tay-Sachs
    disease, Duchenne muscular dystrophy,
    Huntington's chorea, cystic fibrosis, and
    Lesch-Nyhan syndrome are the result of the loss,
    mistaken insertion, or change of a single
    nitrogen base in a DNA molecule.

40
  • 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.
  • Ifand whenthat correct gene begins to function,
    the genetic disorder may be cured. This type of
    procedure is known as human gene therapy (HGT)

41
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.

42
  • 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.

43
GENE THERAPY
44
  • 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.

45
  • 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.

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Types of gene therapy
  • Gene therapy may be classified into the two
    following types
  • Germ line gene therapy
  • Somatic gene therapy

48
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.

49
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.

50
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.

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ETHICS
54
DESIGNER BABIES
55
Vectors in gene therapy
56
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.

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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.

59
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.

60
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.

61
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.

62
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.

63
Physical Methods to Enhance Delivery
64
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.

65
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.

66
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.

67
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.

68
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.

69
  • 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.

70
  • 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.

71
  • What are the ethical issues surrounding gene
    therapy?
  • Because gene therapy involves making changes to
    the bodys 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?

72
PROS OF GENETIC ENGINEERING
73
  • 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.

74
  • Engineered seeds are resistant to pests and can
    survive in a relatively harsh climatic
    conditions.
  • The recently identified plant gene known as
    At-DBF2, 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.

75
  • 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.

76
  • 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.

77
  • 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.

78
  • 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.

79
CONS OF GENETIC ENGINEERING
80
  • 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.

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  • 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.

83
  • 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.

84
  • 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.

85
  • 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.
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