• A set of techniques used to isolate, modify, and transfer genes between organisms.
• It involves creating organisms with recombinant DNA: DNA from two different sources / organisms combined. 
  

1. Isolate the gene that codes for the desired protein.

2. Insert gene into a vector to make recombinant DNA.

3. Transformation- insert recombinant DNA into host cells.

4. Identification of successfully transformed host cells using marker genes.

5. Growth / cloning of transformed host cells.

The diagram shows an overview of the process to transform bacterial cells with the human insulin gene.

1. Isolation of the Desired Gene

Several methods can be used:

• Method 1. Cutting out the gene using restriction endonucleases from DNA.

  • Specific sequences are recognised by the restriction endonucleases and the DNA is cut producing a fragment that contains the gene. 

  • Restriction endonucleases cut at specific recognition sites.

  • They can cut to produce blunt ends or sticky ends. Sticky ends are necessary to insert the fragment into the plasmid.

• Method 2. Using reverse transcriptase to make complementary DNA (cDNA) from mRNA.

  • mRNA is easier to extract from a cell as there is more of it and specialised cells will be expressing the desired gene so will be producing the mRNA.
  • The cDNA contains no introns but sticky ends have to be added after.

• Method 3. Synthesising the gene chemically in a gene machine.

  • Can produce any sequence of nucleotides, has application for synthetic biology to produce genes that don't exist in nature. 
  • The DNA produced has no introns but sticky ends have to be added after. 

2. Insertion into a Vector

• The gene is inserted into a vector:
  • Common vectors: plasmids or viruses.
• Both the gene and plasmid are cut using the same restriction enzyme to create complementary sticky ends.

• DNA ligase is used to join them together, reforming phosphodiester bonds.

3. Introduction into Host Cells

• The recombinant plasmid is introduced into a bacterial host cell (e.g. E. coli).
• Methods include:
  • Heat shock with calcium ions (makes membrane more permeable)
  • Electroporation (electric pulse to open membrane)
  • Microinjection or viral delivery in eukaryotic cells

4. Identification of Transformed Cells

• Marker genes are used to identify bacteria that took up the recombinant plasmid:
  • Antibiotic resistance genes
    • The plasmid contains 2 genes for antibiotic resistance. The target gene is inserted into the middle of the 2nd resistance gene, disrupting it.
    • Cells that have taken up the recombinant plasmid will be resistant to antibiotic 1 but not antibiotic 2.
    • Cells that have taken up the plasmid, but the target gene has not been introduced successfully, will be resistant to both antibiotics.
    • Cells that have not taken up the plasmid will not be resistant to either antibiotic.
• Other types of marker genes may be used such as: 
  • Fluorescent markers
  • Enzyme-based markers

You can learn more about marker genes in a separate study note 'the use of marker genes'.

5. Cloning of Transformed Cells

• Cells that have successfully taken up the recombinant DNA are grown in culture to produce many copies.

• They are grown in optimum conditions.

• Remember bacterial cells use asexual reproduction called binary fission so all cells produced will be genetically identical and will therefore contain the new gene.

• The number of bacteria can be calculated using the formula 2ⁿ where n = the number of divisions.