Chapter%2020%20Biotechnology

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Chapter 20Biotechnology
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Focus of Chapter

• An introduction to the methods and developments
  in
• Recombinant DNA
• Genetic Engineering
• Biotechnology

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Recombinant DNA

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

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Genetic Engineering

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

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Biotechnology

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

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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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Plasmids

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

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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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Insertion

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

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Transformation

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

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Methods

• Temperature shock salt treatment
• Electric current
• Injection

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Identification

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

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Example Applications

• 1. Insulin
• 2. Human Growth Hormone
• 3. Other Proteins

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Comment

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

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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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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

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Nucleic Acid Probes

• Used to find a specific DNA sequence in a mixture
  of DNA pieces

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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.

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Probe Diagram
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DNA Microarray
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PCR

• Polymerase Chain Reaction
• Method for making many copies of a specific
  segment of DNA.
• Also called DNA Amplification.

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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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Importance - PCR

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

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Gel Electrophoresis

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

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RFLP Analysis

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

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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.

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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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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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DNA Sequencers
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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)

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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.

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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.

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Cloning in Plants
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Cloning in Animals

• Has been done by nuclear transplantation.
• Examples Dolly
• Many other vertebrates have now been cloned.

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Dolly Picture
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Cloned cows anyone?
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Stem Cells

• Stem Cell an unspecialized cell that reproduce
  itself or differentiate into other cells.

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Types of Stem Cells

• Embryonic from an embryo.
• Adult found in various tissues of the adult
  body.

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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.

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DNA Technology Applications

• 1. Basic Research
• 2. Medical
• 3. Forensics
• 4. Agricultural

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Basic Research

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

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Assignments

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

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Medical Uses

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

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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.

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Gene Therapy
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PharmAnimals
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Pharmaceutical Products

• Using genetic engineering to produce medical
  proteins.
• Ex. Humilin, HGH, Factor VIII and Factor IX for
  blood clotting, clotting busting proteins etc.

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Forensic Uses

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

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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.

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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.

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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.

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Agricultural Uses

• 1. Animals
• Increased milk production
• Increased feed utilization
• Increased meat production

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Agricultural Uses

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

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Golden Rice
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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

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GMO commercial applications
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Future Of DNA Technology

• Cloning of higher animals.
• Stem Cells - growth of replacement tissues and
  organs.
• Gene therapy to correct DNA defects.
• ?

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Summary

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

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Summary

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

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Questions?

• Viruses?
• Bioethics?