DNA Technology

1
Chapter 20

• DNA Technology

2
Potential uses for cloned genes

• to produce a protein product
• plasminogen activator
• to endow an organism with a metabolic capability
• engineered bacteria that degrade oil spills
• create more copies of the gene for further study

3
Stages of Genetic Engineering

• 1. Isolate desired gene
• 2. Cleavage
• 3. Recombinant DNA
• 4. Clone
• 5. Screen
• 6. Harvest

4
Isolate Desired Gene

• Isolate the desired gene from the genome
• the desired gene will be cut with restriction
  endonucleases (enzymes)

5
Cleavage

• cut DNA of host with restriction endonucleases
  (enzymes)
• several hundred REs exist discovered 1960s
• produce sticky ends or blunt ends

6
Cleavage, cont.

• sticky ends
• Escherichia coli RY13
• Eco RI
• GAATTC
• CTTAAG

• blunt ends
• Haemophilus aegyptius
• Hae III
• GGCC
• CCGG

7
Recombinant DNA

• splice desired gene into host DNA, DNA ligase
  seals the strands
• vector
• plasmid of bacteria (prok) or yeast (euk)
• viral DNA

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Clone

• produce a cell line in which all members have
  identical copies of a particular gene

9
Screen

• Choose cells that carry desired gene
• eliminate those cells that do not carry desired
  gene

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Harvest or Isolate

• harvest protein
• harvest gene

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Harvest protein

• allow culture to produce the gene product
• then collect the polypeptide

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Harvest gene

• remove multiple copies of desired gene from
  culture
• desired gene is then inserted into recipient

13
Sources of genes for cloning

• Directly from an organism
• complementary DNA (cDNA)
• made from mRNA template through reverse
  transcription

14
Alternatives to using Vectors

• Seminatural methods
• protoplast fusion
• electric current technique (electroportation)
• tungsten bullet, DNA gun
• microinjection

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Recombinant-DNA Technology examples

• Interferon
• formerly acquired from live donors
• insulin
• first approved (1982)
• formerly acquired from pancreas slaughtered swine
  and cattle

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Recombinant-DNA Technology examples cont.

• glowing tobacco plant
• plant with firefly glowing gene
• pesticide-resistant crops
• wheat, tomatoes, tobacco, soybeans

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Recombinant-DNA Technology examples, cont.

• bovine somatotropin (growth hormone)
• injected into dairy cattle
• injected into beef cattle
• Snomax?
• snow freezes at higher temp.

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Recombinant-DNA Technology examples, cont.

• human growth hormone
• 2nd approved
• used to treat stunted growth
• vaccines
• genetically engineered produce
• potato, onion

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Human Genome Project

• Collaborative effort to map and sequence entire
  human genome
• 4 approaches
• genetic (linkage) mapping
• physical mapping
• sequencing
• analyzing the genomes of other species

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genetic (linkage) mapping of the human genome

• to locate at least 3000 genetic markers spaced
  evenly throughout the chromosomes
• to make it easier to find other loci

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physical mapping of the human genome

• cutting chromosomes into identifiable fragments
  then determining their order on the chromosome

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sequencing the human genome

• determining the exact nucleotide pairs
• haploid set of human chromosomes contains
  approximately 3 billion nucleotide pairs
• most time-consuming part

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analyzing the genomes of other species

• Analyze genomes of other important species to
  genetic engineering
• E.coli, yeast, mouse, Arabadopsis (a little weed
  helping botanist answer questions about plant
  development)

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Ethical Issues

• Should we engineer new genotypes to individuals
  with anomalies?
• diabetes, CF, immune deficiencies, MD, stunted
  growth
• sickle-cell disease, myopia, altering
  personalities, increase length of life

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• Should we engineer human germ cells?
• If they are carrying abnormal genes
• eugenics - deliberate effort to control the
  genetic makeup of human populations

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• The END