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Chapter%2020%20Biotechnology

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Title: Chapter%2020%20Biotechnology


1
Chapter 20Biotechnology
2
Focus of Chapter
  • An introduction to the methods and developments
    in
  • Recombinant DNA
  • Genetic Engineering
  • Biotechnology

3
Recombinant DNA
  • DNA in which genes from different sources are
    linked.
  • Ex the green mice

4
Genetic Engineering
  • The direct manipulation of genes for practical
    purposes.
  • Ex Using E. coli to produce human insulin.

5
Biotechnology
  • The use of living organisms or their components
    to perform practical tasks.
  • Ex the use of bacteria to digest oil spills.

6
Restrictive Enzymes
  • Cut DNA at specific nucleotide sequences called
    restriction sites.
  • Used to "cut and splice" DNA.
  • Obtained from bacteria.
  • Ex. EcoRI and Hind III

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8
Plasmids
  • Used extensively in Biotechnology and Recombinant
    DNA.
  • Serve as a vehicle for transporting genes.
  • Comment other vehicles are used in other
    methods

9
Steps for Plasmid Use
  • 1. Get the DNA for the trait.
  • 2. Insert DNA into the plasmid.
  • 3. Bacterial Transformation.
  • 4. Identification of the new trait.

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11
Insertion
  • Placing foreign DNA into a plasmid.
  • Open plasmid with enzymes to create sticky
    ends.
  • Splice the new DNA and plasmid together.

12
Transformation
  • Placing the plasmid into a bacterial cell.
  • Reminder - our lab need to know for AP testing
    purposes.

13
Methods
  • Temperature shock salt treatment
  • Electric current
  • Injection

14
Identification
  • Screening the altered cells for the desired gene.
  • Ex Antibiotic sensitivity or the expression of
    a new trait (color, glowing etc.).

15
Example Applications
  • 1. Insulin
  • 2. Human Growth Hormone
  • 3. Other Proteins

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17
Comment
  • Gene cant be above a certain size (12 kb) or a
    plasmid wont work.
  • mRNA must not need splicing to remove introns.

18
DNA Sources
  • 1. Organism - use a section of their chromosome.
  • 2. cDNA - Complementary DNA - created copy of
    DNA from the mRNA transcript to avoid introns.
    Uses reverse transcriptase.

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20
Other Vehicles
  • BACs Bacteria Artificial Chromosome handle
    inserts of 100-300 kb
  • YACs Yeast Artificial Chromosome have a
    centromere and telomomere, handle inserts gt300 kb
  • HACs Human Artificial Chromosome

21
Nucleic Acid Probes
  • Used to find a specific DNA sequence in a mixture
    of DNA pieces

22
How Used
  • DNA is denatured and cut to produced ss pieces.
  • Piece of compliment DNA is added as a probe.
    The probe has been labeled.
  • Look for where the probe goes in the DNA sample.

23
Probe Diagram
24
DNA Microarray
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26
PCR
  • Polymerase Chain Reaction
  • Method for making many copies of a specific
    segment of DNA.
  • Also called DNA Amplification.

27
PCR - Method
  • 1. Separate strands by heating (denature the
    DNA).
  • 2. Cool slightly.
  • 3. Build new strand from primers and nucleotides.
  • 4. Repeat.

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29
Importance - PCR
  • Can amplify any DNA with as little as one
    original copy.
  • Very useful in a variety of techniques and tests.

30
Gel Electrophoresis
  • Technique will be covered in lab.
  • Used to separate mixtures of DNA or proteins.

31
RFLP Analysis
  • Restriction Fragment Length Polymophisms.
  • Method for detecting minor differences in DNA
    structure between individuals.
  • Common in DNA fingerprinting

32
Method
  • 1. Digest DNA with restrictive enzymes.
  • 2. Separate pieces by Gel Electrophoresis
  • 3. Identify sequences with probes.
  • 4. May use PCR to amplify specific variable
    regions of the DNA.

33
RFLP - Results
  • Patterns of DNA markers or DNA fingerprint
  • Markers are inherited in a Mendelian pattern and
    can show relationships (Pedigree studies).

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36
DNA Sequencing
  • Uses dideoxynucleotides which stop DNA Polymerase
    at a known point in DNA replication.
  • Builds new DNA from single strand DNA.
  • Produces fragments of different lengths.

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38
DNA Sequencers
39
Application
  • DNA sequence is read base by base.
  • By sequencing overlapping pieces of chromosomes,
    the entire genome of an organism can be read.
    (chromosome walking)

40
Genome Sequencing
  • The DNA sequence has been read for the
    following
  • 4,000 bacteria species
  • 190 archaeal species (a type of prokaryote)
  • 180 eukaryotic species
  • More than 17,000 species are in progress
  • Includes several cancers, ancient humans and
    bacteria that live in our gut.

41
Cloning of Organisms
  • Reproducing an organisms by asexual means.
  • Commonly done in plants.
  • Shows the concept of Totipotency that a
    single cell can develop into a new organism.

42
Cloning in Plants
43
Cloning in Animals
  • Has been done by nuclear transplantation.
  • Examples Dolly
  • Many other vertebrates have now been cloned.

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45
Dolly Picture
46
Cloned cows anyone?
47
Stem Cells
  • Stem Cell an unspecialized cell that reproduce
    itself or differentiate into other cells.

48
Types of Stem Cells
  • Embryonic from an embryo.
  • Adult found in various tissues of the adult
    body.

49
Research
  • Attempting to use stem cells to replace damaged
    cells or body parts.
  • If can use own stem cells, avoids tissue
    rejection problems. Ex grow a new
    bladder.

50
DNA Technology Applications
  • 1. Basic Research
  • 2. Medical
  • 3. Forensics
  • 4. Agricultural

51
Basic Research
  • 1. DNA and protein studies
  • 2. Evolution
  • 3. Gene structure and control mechanisms.

52
Assignments
  • Read Chapter 20, Chapter 13 in Hillis
  • Chapter 19 before spring break
  • Chapter 20 Wed. 3/19
  • Exam 2 Tuesday 3/25

53
Medical Uses
  • 1. Diagnosis of Diseases
  • 2. Gene Therapy
  • 3. Vaccines
  • 4. Pharmaceutical Products

54
Disease Identification
  • Knock-out mice engineered to knock-out one or
    both copies of a gene in order to determine the
    function of the gene.
  • Very BIG study technique in medicine and disease
    studies.
  • Model organism for understanding the disease at
    the molecular level and for testing medicines and
    treatments.

55
Gene Therapy
56
PharmAnimals
57
Pharmaceutical Products
  • Using genetic engineering to produce medical
    proteins.
  • Ex. Humilin, HGH, Factor VIII and Factor IX for
    blood clotting, clotting busting proteins etc.

58
Forensic Uses
  • DNA fingerprints for crime solving used in
    every TV crime show
  • DNA identification records standard for the
    military

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60
Paternity Cases
  • Variable regions of DNA are cut out and amplified
    by PCR.
  • Often used tandem repeat regions (DNA sections
    that repeat).
  • Look at sets of tandem repeat areas with known
    frequencies.
  • Any mismatch excludes the father.
  • Consistent matches keen the individual as a
    candidate.

61
Current uses or applications
  • Some pedigree registrations now require a proof
    by a match of the DNA of the offspring to the
    sire and dam on the pedigree application.
  • Common in a number of animal pedigree
    registrations such as dogs, horses, beef etc.

62
Comments
  • Links suspect bodily to the crime scene, but
    doesnt prove they committed the crime.
  • Results take MUCH longer than on TV shows.
  • Analysis of old evidence is reversing some
    sentences.

63
Agricultural Uses
  • 1. Animals
  • Increased milk production
  • Increased feed utilization
  • Increased meat production

64
Agricultural Uses
  • 2. Plants
  • Herbicide resistance
  • Retard spoilage of fruits
  • Insect resistance BT corn
  • Nitrogen-Fixation ability

65
Golden Rice
66
Genetically Modified Organism or GMO
  • Produced by direct genetic manipulation, not
    traditional breeding practices.
  • FDA just approved sale of GMO animal products for
    human consumption.
  • Bioethics concerns

67
GMO commercial applications
68
Future Of DNA Technology
  • Cloning of higher animals.
  • Stem Cells - growth of replacement tissues and
    organs.
  • Gene therapy to correct DNA defects.
  • ?

69
Summary
  • Know the basics of some of the DNA technology
    techniques.
  • Know
  • Bacterial transformation lab
  • How Gel electrophoresis works
  • Restriction enzymes

70
Summary
  • Watch the news for DNA technology discoveries.
    Be able to discuss one recent event.

71
Questions?
  • Viruses?
  • Bioethics?
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