Amplifying DNA Fragments

• Gene cloning is all about making loads of identical copies of a gene and can be done using two different techniques: in vivo and in vitro
• In vivo cloning?

  Where the gene copies are made within a living organism. As the organism grows and divides, it replicates the DNA, creating multiple copies of the gene
• In vitro cloning?

  Where the gene copies are made outside of a living organism using the polymerase chain reaction (PCR)
• In Vivo cloning process?

  Once you have the DNA fragment containing the target gene you can use it for in vivo cloning
• In Vivo cloning Part 1 - making recombinant DNA


  1. Insert the DNA fragment into a vector's DNA

  2. Isolate the vector DNA

  3. Use restriction endonucleases and DNA ligase to stick the DNA fragment and vector DNA together
• Vector DNA


  DNA used as a carrier to insert the target DNA fragment
• Cutting open vector DNA

  1. Using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene

  2. Means the sticky ends of the vector DNA are complementary to the sticky ends of the DNA fragment containing the gene
• Mixing vector DNA and DNA fragment

  1. With DNA ligase

  2. Joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA - ligation
• Recombinant DNA

  New combination of bases in the DNA (vector DNA + DNA fragment)
• What is a vector?

  something that is used to transfer DNA into a cell. Can be plasmids (small circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria)
• Part 2 - transforming cells

  The vector with the recombinant DNA is used to transfer the gene into cells (host cells) that take up the vectors containing the gene of interest and are said to be transformed. If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA
• e.g. of transforming cells?

  host bacterial cells are placed into ice-cold calcium chloride solution to make their cell walls more permeable. The plasmids are added and the mixture is heat-shocked (heated to around 42 degrees for 1-2 mins), which encourages the cells to take in the plasmids
• With a bacteriophage vector, the bacteriophage will infect host bacterium by injecting its DNA into it. The phage DNA (with the target gene in it) then integrates into the bacterial DNA