8.18 Recombinant DNA

• The genetic code is the basis for storing instructions that, alongside environmental influences, dictate the characteristics of organisms
• The genetic code is universal, meaning that almost every organism uses the same four nitrogenous bases A, T, C, and G 
  • The universal nature of the genetic code means that the same codons code for the same amino acids in all living things; genetic information is therefore transferable between species
  • The mechanisms of transcription and translation are also universal which means that the transferred DNA can be translated within cells of another species
• Scientists can artificially change an organism's DNA by combining lengths of nucleotides from different sources; typically the nucleotides are from different species
• The altered DNA, with the introduced nucleotides, is called recombinant DNA 
• If an organism contains nucleotide sequences from a different species it is known as a transgenic organism or a genetically modified organism (GMO)

Transferring genes from bacteria into the DNA of maize plants creates recombinant DNA

• Producing a transgenic organism involves the following process
  • Identification of the desired gene
    • This gene will code for a desired characteristic, e.g.
      • Pest resistance genes in crops 
      • The human insulin gene 
  • Isolation of the desired gene by, e.g.
    • Using an enzyme called reverse transcriptase to convert a desired length of mRNA back into DNA; DNA produced in this way is known as complementary DNA, or cDNA
    • Cutting the gene from its location on a chromosome using enzymes called restriction endonucleases
    • Designing and building synthetic DNA sequences in a lab
  • Multiplication of the gene, i.e. producing many copies, or clones; this can be carried out using the polymerase chain reaction (PCR)
    • PCR machines known as thermocyclers use free nucleotides, DNA polymerase, and DNA primers to produce many identical copies of a desired gene
  • Transfer of the desired gene into another organism's DNA using a vector, e.g. DNA plasmids, viruses, or fatty envelopes known as liposomes
    • Once another organism has taken up the vector it is said to be transformed
  • Identification of the cells that contain the new gene by using a marker gene alongside the desired gene; this means that any cells that take up the desired gene will take up the marker gene as well e.g.
    • Antibiotic resistance; transformed cells will survive if treated with a specific antibiotic
    • Fluorescence; transformed bacterial cells will fluoresce under UV light
• Once the transformed cells have been identified they can be cloned, ensuring that all new cells contain copies of the desired gene
  • In the case of bacteria this can be carried out in a large container known as a fermenter

DNA can be transferred from one organism to another to produce recombinant DNA; this process involves identification, isolation, multiplication, and transfer of the desired gene, followed by identification and cloning of the transformed organisms

Isolating the desired gene using restriction endonucleases

• Restriction endonucleases are enzymes that cut DNA
  • They are sometimes referred to as restriction enzymes 
• There are many different restriction endonucleases, each of which binds to a specific sequence of bases known as a restriction site on DNA, e.g. the restriction endonuclease HindIII will always bind to the base sequence AAGCTT
• Restriction endonucleases separate DNA at restriction sites by cutting the sugar-phosphate backbone in an uneven way; this leaves exposed single-stranded sequences of bases known as 'sticky ends'
  • Sticky ends result in one strand of the DNA fragment being longer than the other strand
• The sticky ends make it easier to insert the desired gene into another organism's DNA or into vector DNA as they can easily form hydrogen bonds with complementary base sequences that have been cut with the same restriction endonucleases

Restriction enzymes produce a jagged cut at a restriction site, leaving 'sticky ends'

Inserting the desired gene into a vector using DNA ligase

• Once the desired gene has been cut from DNA using the relevant restriction endonuclease, it can then be transferred into the DNA of a vector, e.g.
  • A DNA plasmid
  • A vector organism such as a virus or bacterium
• The DNA of the vector will be cut using the same restriction endonuclease as the desired gene, leaving complementary sticky ends
• The enzyme DNA ligase is used to catalyse the formation of phosphodiester bonds between the sugar-phosphate backbone of the desired gene and that of the vector DNA
  • If this is carried out using a plasmid, the plasmid will be known as a recombinant plasmid

DNA ligase is used to join the isolated gene to the vector DNA