Chapter 16 Recombination DNA and Genetic Engineering

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Chapter 16 Recombination DNA and Genetic
Engineering
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Cloning DNA

• 1. Cloning is the production of identical copies
• 2. An underground stem sends up new shoots that
  are clones
• 3. Members of a bacterial colony on a petri dish
  are clones because
• they all came from division of the same cell.
• 4. Human identical twins are clones the original
  single embryo separate to become two individuals.
• 5. Gene cloning is production of many identical
  copies of the same gene.

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• 6. If the inserted gene is replicated and
  expressed, we can recover the cloned gene or
  protein product.
• 7. Cloned genes have many research purposes
  determining the base sequence between normal and
• mutated genes, altering the phenotype, etc.
• 8. Humans can be treated with gene therapy
  alteration of other organisms forms transgenic
  organisms.

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Clogged Arteries

• Cholesterol does good things for the body, such
  as forming membranes and vitamin D, but it can
  also combine with lipoproteins to form
  atherosclerotic plaques in the walls of the
  arteries
• Some persons have genes that cause familial
  cholesterolemia
• Gene therapy promises a way to genetically alter
  the cells of the liver to keep the levels of
  cholesterol in the more normal range

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• We can engineer genetic changes through
  recombinant DNA technology.
• DNA from different species can be cut, spliced
  together, and inserted into bacteria, which then
  multiply the DNA necessary for protein production
• Genetic engineering has great promise for
  agriculture, medicine, and industry.

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Restriction Enzyme

• Bacteria possess restriction enzyme whose usual
  function is to cut apart foreign DNA molecules.
• Each enzyme cut only at sites that possess a
  specific base sequence
• The wide variety of restriction enzymes and their
  specificity makes it possible to study the genome
  of a particular species

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Modification Enzymes

• Many times the sticky ends that result from the
  cut can be used to pair up with another DNA
  fragment cut by the same enzyme.
• DNA fragments produced by restriction enzymes are
  treated with DNA ligase to splice the DNA
  fragments together to form a recombinant DNA
  molecule.

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Cloning Vectors for Amplifying DNA

• Plasmids are circular DNA molecules in bacteria
  that carry only a few genes and can replicate
  independently of the single main chromosomes
• When the plasmids is replicated, any foreign DNA
  that might have become incorporated into it is
  also replicated producing a DNA clone.

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• Modified plasmids that are capable of accepting,
  replicating, and delivering DNA to another host
  cell are called cloning vectors

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Reverse Transcriptase to Make cDNA

• Even after a desired gene has been isolated and
  amplified, it may not be translated into
  functional protein by the bacteria because
  introns (noncoding regions) are still present.
• Researchers minimize this problem by using cDNA,
  which is made from mature mRNA transcripts.

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• The cDNA is made from mRNA by reverse
  transcriptase
• cDNA can be inserted into a plasmid for
  amplification

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PCR

• The polymerase chain reaction (PCR) can be used
  to make millions of copies of cDNA

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• 1. PCR can create millions of copies of a single
  gene or a specific piece of DNA in a test tube.
• 2. PCR is very specificthe targeted DNA sequence
  can be less than one part in a million of the
  total DNA sample therefore a
• single gene can be amplified using PCR.
• 3. The polymerase chain reaction (PCR) uses the
  enzyme DNA polymerase to carry out multiple
  replications (a chain reaction) of
• target DNA.
• 4. PCR automation is possible because
  heat-resistant DNA polymerase from Thermus
  aquaticus, which grows in hot springs, is an
  enzyme that withstands the temperature necessary
  to separate double stranded DNA.

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What are Primers?

• Primers are short nucleotide sequences that are
  made in the laboratory.
• They are recognized by DNA polyermases as the
  START tags for building complementary sequences
  of DNA dictated by computer programs stored in
  the machines.
• DNA FINGERPRINTS READ PAGE 257

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How is DNA sequenced?

• Current laboratories use automated DNA sequencing
  to determine the unknown sequence of bases in a
  DNA sample.
• The automated DNA sequencer separates the sets of
  fragments by gel electrophoresis.
• The tag base at the end of each fragment in the
  set is identified by laser beam.
• The computer program in the machine assembles the
  information from all the nucleotides in the
  sample to reveal the entire DNA sequence

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How can you isolate a particular gene for study?

• Gene Library a collection of bacteria that house
  different cloned DNA fragments
• The library of the entire genome or of cDNA is
  free of introns

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What are Probes?

• DNA probes, short DNA sequences assembled from
  radioactive nucleotides, can pair with parts of
  the gene to be studied
• This nucleic acid hybridization technique can be
  used with other procedures to select cells and
  their DNA, which may be of interest to the
  researcher

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Analyzing DNA Segments

• 1. Mitochondria DNA sequences in modern living
  populations can decipher the evolutionary history
  of human populations.
• 2. DNA fingerprinting is the technique of using
  DNA fragment lengths, resulting from restriction
  enzyme cleavage and amplified
• by PCR, to identify particular individuals.
• a. DNA is treated with restriction enzymes to cut
  it into different sized fragments.
• b. During gel electrophoresis, fragments separate
  according to length, resulting in a pattern of
  bands.
• .

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• c. DNA fingerprinting can identify deceased
  individuals from skeletal remains, perpetrators
  of crimes from blood or semen
• samples, and genetic makeup of long-dead
  individuals or extinct organisms

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• 3. PCR amplification and DNA analysis is used to
• a. detect viral infections, genetic disorders,
  and
• cancer
• b. determine the nucleotide sequence of human
• genes the Human Genome Project and
• c. associate samples with DNA of parents because
  it is inherited.

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Using the Genetic Strips

• Microorganisms can produce useful substances such
  as human insulin and blood-clotting factors.
• Genetically engineered bacteria can clean up
  messes such as oil spills
• Gene may help use devise counterattacks against
  rapidly mutating pathogens

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Transgenic Bacteria

• 1. Bacteria are grown in large vats called
  bioreactors.
• a. Foreign genes are inserted and the product is
  harvested. (insulin, hepatitis B vaccine, t-PA,
  and human growth hormone)
• 2. Transgenic bacteria have been produced to
  protect and improve the health of plants (frost
  resistance, pesticides)
• 3. Transgenic bacteria can degrade substances
  (oil-eating bacteria, bio-filters)
• 4. Transgenic bacteria can produce chemical
  products. (phenylalanine for aspartame sweetener)

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Designer Plants (Transgenic Plants)

• Regenerating plants from cultured cells
• Botanist are searching the world for seeds from
  the wild ancestors of potatoes, corn, etc.
• They are worry is that there is too little
  diversity in the few strains now used for food
  crops
• Many plant species can be regenerated from
  cultured cells.

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• Useful mutations, such as resistance to a toxin,
  can be identified
• Foreign genes now give cotton, corn, and potato
  strains ability to produce an insect toxin
• Plants are being engineered to produce human
  proteins including hormones, clotting factors,
  and antibodies in their seeds antibodies made by
  corn deliver radioisotopes to tumor cells and a
  soybean engineered antibody can treat genital
  herpes.

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How are Genes Transferred Into Plants?

• An early experiment showed that a plasmid from a
  bacterium that normally causes tumors in plants
  could be modified by replacing the tumor gene
  with the desirable genes

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Gene Transfer in Animals

• Supermice and Biotech Barnyards
• In 1982, the rat gene for somatotropin production
  was introduced into mouse eggs the mice which
  subsequently expressed the rat gene grew larger
  than their littermates
• Farm animals may be used to produce TPA for
  diminishing the severity of heart attacks or CFTR
  is in the diminishing of cystic fibrous

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• 1. Animal use requires methods to insert genes
  into eggs of animals.
• a. It is possible to micro-inject foreign genes
  into eggs by hand.
• b. Vortex mixing places eggs in an agitator with
  DNA and silicon-carbide needles that make tiny
  holes which the DNA can enter.
• c. Using this technique, many types of animal
  eggs have been injected with bovine growth
  hormone (bGH) to produce larger fishes, cows,
  pigs, rabbits, and sheep.

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Gene Pharming

• Gene pharming is the use of transgenic farm
  animals to produce pharmaceuticals obtainable
  from the milk of females.
• a. Genes for therapeutic proteins are inserted
  into animals DNA animals milk produces
  proteins.
• b. Drugs obtained through gene pharming are
  planned for the treatment of cystic fibrosis,
  cancer,blood diseases, and other disorders.

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Cloning Transgenic Animals

• 1. For many years, it was believed that adult
  vertebrate animals could not be cloned the
  cloning of Dolly in 1997 demonstrated this can be
  cone.
• 2. Cloning of an adult vertebrate would require
  that all genes of an adult cell be turned on
  again.

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• 3. Cloning of mammals involves injecting a 2n
  nucleus adult cell into an enucleated egg.
• 4. The cloned eggs begin development in vitro and
  are then returned to host mothers until the
  clones are born.
• 5. Somatic Cell Nuclear Transfer cloning from
  adult cells (Dolly)

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Animal Organs as Biotechnology Products

• 1. It may be possible to use genetically
  engineered pigs to serve as a source of organs
  for human transplant.
• 2. Scientists are learning how to stimulate human
  cells to construct organs in the laboratory.

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Mapping the Human Genome

• Human Genome Initiative is dependent on
  technology
• The information gained will give insights into
  genetic disorders and utimately provide gene
  therapy
• New field of genomics will be concerned with
  mapping and sequencing the genomes as well
  understanding the evolutionary relationships

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Safety Issues

• Genetically Engineered bacteria have fail safe
  genes included in the DNA which are supposed to
  be lethal if the bacteria escapes into a non-lab
  environment
• General public is concerned about organisms being
  released that are not natural and may endanger
  human lives

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Biotechnology in a Brave New World

• Microarray or gene chips can reveal a stunning
  amount of information about an individual DNA

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Gene Therapy

• Has been successful in a trail against SCID-X1
• Eugenic engineering is idea of being able to
  select desirable humans traits

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Send in the Clones

• Xenotransplantation is the transferring of an
  organ from one species to another
• Pigs can be engineered to lack certain genes that
  would cause rejection problems when their organs
  are transplanted to humans

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Weighing the Benefits and Risks

• Some say we should never alter the DNA of any
  organisms others say we already have
• What would be problems with genetic engineering
  over the long haul?

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• Scenario 1 Melissa is a happy 5 year old who is
  loved by her family. She becomes ill and is
  diagnosed with childhood leukemia. A desperate
  search ensues to find a bone marrow donor whose
  type matches Melissa. After a year of searching,
  Melissas outlook is grim. Her family decides to
  clone Melissa so that her clone could be the bone
  marrow donor. Do you think this is a god idea?
  Why or why not.

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• Scenario 2 A well-loved horse named Barbero
  breaks his leg in a race. Many people were
  praying for his well being and thousands of
  dollars were spent trying to get him to recover.
  Mail and flowers poured into the animal hospital
  and stable where Barbero lived. Also, after a
  year of poor recovery, the decision was made to
  euthanize Barbero. The owners save sample of
  his DNA so that Barbero can be cloned. Do you
  think they should clone him? Why or why not.