DNA fragments

• Recombinant DNA technology


  Recombining or putting together two different organisms' DNA
• Purpose of recombinant DNA technology


  • For industrial processes and medical treatments
• Steps in recombinant DNA technology

  1. Isolate gene of interest

  2. Clone gene to make many copies

  3. Insert gene into another organism
• Reverse transcription


  Using the enzyme reverse transcriptase to make DNA copies from mRNA
• Organisms with reverse transcriptase

  • Viruses like HIV
• Reverse transcription process


  1. Extract mRNA from cell producing protein of interest

  2. Mix with complementary DNA nucleotides

  3. Add reverse transcriptase enzyme to catalyze joining of nucleotides

  4. Can then add DNA polymerase to make double-stranded DNA
• Advantages of reverse transcription

  • Introns are already removed from mRNA, so resulting DNA is intron-free
• Restriction endonucleases


  Enzymes that cut DNA, found naturally in bacteria as a defense mechanism
• Characteristics of restriction endonucleases


  • Different enzymes have different recognition sequences where they cut DNA
  • Some cut at palindromic sequences to create sticky ends
• Gene machine


  Computerized machine used in the lab to synthesize DNA fragments
• Gene machine process


  1. Determine amino acid sequence of desired protein

  2. Work out DNA sequence that codes for those amino acids

  3. Computer checks sequence for safety and ethics

  4. Computer synthesizes small overlapping DNA fragments (oligonucleotides)

  5. Oligonucleotides are joined to create full DNA fragment

  6. DNA fragment can then be amplified using PCR
• Comparison of DNA fragment isolation methods

  • Reverse transcription
  • Restriction endonucleases
  • Gene machine
• Pros and cons of reverse transcription

  • Pros: Can use cells naturally producing protein to get lots of mRNA

  Cons: More time-consuming and technically difficult
• Pros and cons of restriction endonucleases

  • Pros: Can select enzymes to create sticky ends for easier insertion

  Cons: Original DNA still contains introns
• Pros and cons of gene machine

  • Pros: Very quick, accurate, can design DNA fragment exactly, no introns

  Cons: Need to determine amino acid and DNA sequence first