Gene technology

• Recombinant DNA

  combination of DNA from two different organisms 
• Transgenic

  Organism that contains DNA from two different organisms 
• Where is the enzyme reverse transcriptase found?

  Retroviruses
• What does reverse transcriptase do?

  Turns viral RNA into DNA so that it can be transcribed by the host cells into proteins
• How is reverse transcriptase used to copy the insulin gene?

  1. Extract mature mRNA coding for insulin
  2. Single stranded complimentary copy of DNA (cDNA) is formed using reverse transcriptase on the mRNA template
  3. cDNA is used to form double stranded DNA using DNA polymerase
  4. Forms a double stranded copy of the human insulin gene
• Why do bacteria contain restriction endonucleases?

  To protect themselves from viruses
• What do restriction endonucleases do?

  Cut viral DNA up at specific sites
• What active site do restriction endonucelases have?

  Highly specific
• Where do restriction endonucleases cut?

  Recognition sites (usually 4-8 base pairs long)
• What is special about recognition sites?

  They are usually palindromic
• What are examples of restriction endonucleases?

  • EcoRI
  • HindIII
• Cut sites

  A) Sticky ends

  B) Blunt ends

  
• What are sticky ends?

  Single-stranded overhangs that bind with another sticky end that has been cut by the same restrictive endonucleases
• What is a gene machine?

  Can synthesise DNA from scratch without a DNA template using a database
• What is the method for gene machines?

  1. Sequence is designed
  2. 1st nucleotide is fixed to a support bead
  3. Nucleotides are attached 1 by 1, in the correct order (they are protected to prevent branching)
  4. Oligonucleotides (20 nucleotides long) are produced
  5. Broken off the support and protection is removed
  6. Oligonucleotides can be joined together to make longer DNA fragments
• What does in vivo mean?

  In living
• What does in vitro mean?

  In glass