Recombinant DNA

• Recombinant DNA technology


  The transfer of DNA fragments from one organism to another (Or species)
• Recombinant DNA technology works as

  1. The genetic code is universal

  2. Therefore transcription & translation occur by the same mechanism & result in the same amino
• Methods to produce fragments of DNA


  • Conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase
  • Using restriction enzymes to cut a fragment containing the desired gene from DNA
  • Creating the gene in a 'gene machine'
• Using reverse transcriptase to produce DNA fragments

  1. mRNA complementary to the target gene is used as a template

  2. It's mixed with free nucleotides which match up to their base pairs, & reverse transcriptase which forms the sugar- phosphate backbone, to create cDNA (complementary DNA)
• Using endonucleases to cut DNA fragments

  1. Restriction endonucleases (RE) cut DNA at specific sequences

  2. Different REs cut at different points but 1 RE will always cut at the same sequence

  3. Therefore using particular REs allows you to cut out a certain gene of interest
• Ways to amplify DNA fragments

  • In Vitro / polymerase chain reaction (PCR)
  • In-vivo gene cloning
• Reaction mixture in the first stage of PCR- In-Vitro

  1. Contains the DNA fragment to be amplified

  2. Primers that are complementary to the start of the fragment

  3. Free nucleotides to match up to exposed bases

  4. DNA polymerase to create the new DNA
• Process of amplifying DNA fragments using PCR


  1. DNA heated (95) to denature DNA & break hydrogen bonds & separate DNA strand

  2. Cooled (55) to allow primers to bind (to complementary base sequences)

  3. Heated again (70) to activate DNA polymerase (form phosphodiester bonds) & allow free nucleotides to join

  4. New DNA acts as template for next cycle
• Process of inserting a DNA fragment into a vector


  1. A plasmid (circular DNA from bacteria) is used as the vector, & is cut using the same restriction enzymes as the DNA, so that the ends are complementary

  2. DNA ligase joins the fragment & plasmid together
• Process of inserting a vector into a host cell (cell transformation)

  1. Host cells (bacteria) are mixed with the vectors in an ice-cold solution, then heat shocked to encourage the cells to take up the vectors

  2. The cells can then be grown & the DNA fragment will be cloned
• Identifying transformed cells


  Marker genes e.g. fluorescence, antibiotic resistant genes & enzyme markers can also be inserted into vectors along with the DNA. When cells begin to grow, UV light can be used to identify which cells have taken up the vector & which haven't. Those that are not fluorescent mean the bacteria have successfully taken up the vector