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HBIO4 > Genetic Engineering
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Recombinant DNA

  • DNA probe
    • Used to locate genes
    • Single-stranded DNA that is complementary to a specific gene
    • Labeled with radioisotope or fluorescent dye
    • Allows location of probe when it binds to genes
  • Gene isolation
    • 2 possible mechanisms: restriction enzymes or reverse transcriptase
    • Restriction enzymes
      • Endonucleases cut DNA at specific recognition sites by hydrolysis
      • Hydrogen bonds break
      • Leaving sticky ends
    • Reverse transcriptase
      • Taken from a retrovirus
      • Catalyses the formation of single-stranded DNA from mRNA
  • Polymerase chain reaction
    • Produces larger quantities of DNA
    • Hydrogen bonds of double-stranded DNA are broken by applying heat
    • Primers are added to mark the START and END of the gene to be copied
    • Nucleotides and DNA polymerase are added
    • Enzyme attaches nucleotides to single stranded DNA
    • Produces two double-stranded DNA molecules
    • Procedure is repeated until enough DNA is produced
  • Transfer of recombinant DNA
    • Vector is a gene carrier → carries a human gene into the cell of a bacterium or yeast
    • Plasmid
      • Circular strand of DNA found in bacteria
      • Useful vector to make human protein from bacteria
    • Plasmid DNA is cut by using restriction enzymes
    • Recombinant gene is inserted into plasmid by DNA ligase
    • Plasmid is introduced into bacteria that do not contain any plasmids
  • Genetic markers
    • Only 1% of bacteria take up the engineered plasmid
    • Identify by using antibiotic resistance as a genetic marker
    • Gene for antibiotic resistance is added next to the recombinant gene in the plasmid
    • Antibiotic is added to culture → only genetically modified bacteria survive

Production of Human Insulin

  • Isolate human gene for insulin by using cytoplasmic mRNA (no introns)
  • Reverse transcriptase produces DNA from mRNA
  • DNA produced is given "sticky ends"
  • Insert into bacterial plasmid
    • Cell wall is dissolved using enzymes
    • Centrifuge separates bacterial chromosome ring from plasmids
    • Cut open the plasmid with restriction enzymes
  • Mix plasmid and DNA and join them with DNA ligase
  • Add antibiotic resistance gene next to insulin gene in plasmid
  • Add antibiotic to culture
  • Grow transformed cells using industrial fermenters
  • Isolate and purify human protein made by these cells

Genetically modified organisms

Selective breeding

  • Takes longer than genetic engineering
  • Involves sexual reproduction (non-specific)
    • Offspring have new combination of alleles
    • May develop an unwanted characteristic
    • More susceptible to disease, more aggressive
  • Genetic engineering involves the transfer of one specific gene

Moral and ethical issues

  • Transgenic bacteria or viruses may mutate and may become pathogenic
  • Genetically modified crops could "escape"
    • Forms a genetically modified population in the environment
    • Genetic modification may involve the resistance to herbicides
    • Escaped crops may become "superweeds" that are difficult to kill and control
  • Transgenic organisms could upset the balance of nature
    • Population of transgenic salmon have been produced in which individuals grow rapidly
    • These transgenic fish could compete for food with other fish species
  • Genetically engineered food
    • May cause unknown disease in humans in the long-term

Genome Project

  • Nucleotide base sequences of human DNA has been determined
    • Not all DNA codes for proteins
      • Non-coding DNA
      • Regulatory genes
      • Telomeres are repetitive sequences of DNA
  • Next task is to determine the proteome
    • Collection of all human proteins
    • Done by studying the genome of simpler organisms
  • Sequences DNA allows
    • Production of vaccine against Plasmodium (malaria)
      • Compare genome of many plasmodium parasites
      • Identify genes which code for antigens
      • Done by looking at genes that have a wide range of variation across parasites
      • Variation is caused by natural selection
      • Indicates that these genes are most likely to be susceptible to the immune system
      • Can be used as targets for vaccines
    • Identification of predisposition to cancer
    • New treatments