New Genes for Old

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