Producing DNA Fragments

• Recombinant DNA technology is combining different organisms' DNA to alter genes to improve industrial processes and medical treatments. First step is isolating DNA fragments.
• Reverse transcription


  1 - enzyme makes DNA copies from MRNA

  2 - naturally occurs in viruses such as HIV

  3 - cells that naturally produce protein of interest selected

  4 - cels have large amount of mRNA for the protein

  5 - reverse transcriptionase joins nucleotides with complementary base pairs in DNA sequence

  6 - single strand of DNA made cDNA (use add more nucleotides and DNA polymerase for double)
• Restriction endonucleases


  1 - cut up DNA

  2 - naturally occur in bacteria

  3 - restriction enzymes have active site complementary to range of DNA base sequences (recognition sequences) so each enzyme cuts DNA at specific location

  4 - some enzymes cut staggered ends so leave DNA bases open to join to complementary base pairs
• Gene machine


  1 - DNA fragments created in lab using computerised machine

  2 - examine protein of interest to identify amino acid sequence

  3 - use that to work out what the mRNA sequence would be

  4 - computer cuts small section of overlapping single strands of nucleotides (oligonucleotides)

  5 - can then be joined together to create DNA for entire gene (use PCR to amplify