gene technologies

• Genetic code


  Universal, meaning that almost every organism uses the same four nitrogenous bases – A, T, C & G
• Genetic code


  The basis for storing instructions that, alongside environmental influences, dictate the behaviour of cells and as a result, the behaviour of the whole organism
• The universal nature of the genetic code means that the same codons code for the same amino acids in all living things (meaning that genetic information is transferable between species)
• Recombinant DNA (rDNA)


  Altered DNA, with the introduced nucleotides from different sources (typically the nucleotides are from different species)
• Transgenic organism


  An organism that contains nucleotide sequences from a different species
• Genetically modified organism (GMO)


  Any organism that has introduced genetic material
• The mechanisms of transcription and translation are also universal which means that the transferred DNA can be translated within cells of the genetically modified organism
• Recombinant DNA technology


  Form of genetic engineering involving the transfer of fragments of DNA from one organism/species into another organism/species
• Genetically engineered organism


  Contains recombinant DNA and is a genetically modified organism (GMO)
• Steps to genetically engineer an organism

  1. Identification of the DNA fragment or gene

  2. Isolation of the desired DNA fragment

  3. Multiplication of the DNA fragment (using polymerase chain reaction - PCR)

  4. Transfer into the organism using a vector (e.g. plasmids, viruses, liposomes)

  5. Identification of the cells with the new DNA fragment (by using a marker), which is then cloned
• What genetic engineers need to modify an organism

  • Enzymes (restriction endonucleases, ligase and reverse transcriptase)
  • Vectors - used to deliver DNA fragments into a cell (eg. plasmids, viruses and liposomes)
  • Markers - genes that code for identifiable substances that can be tracked (eg. GFP - green fluorescent protein which fluoresces under UV light or GUS - β-glucuronidase enzyme which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent)
• Synthetic biology


  New field of science that studies the design and construction of different biological pathways, organisms and devices, as well as the redesigning of existing natural biological systems
• Genetic engineering


  The deliberate modification of a specific characteristic (or characteristics) of an organism
• Genetic engineering technique

  1. Removing a gene (or genes), with the desired characteristic, from one organism

  2. Transferring the gene (using a vector) into another organism

  3. Desired gene is then expressed
• Ways to obtain the gene with the specific characteristic

  • Extracting the gene from the DNA of a donor organism using enzymes (restriction endonucleases)
  • Using reverse transcriptase to synthesise a single strand of complementary DNA (cDNA) from the mRNA of a donor organism
  • Synthesising the gene artificially using nucleotides in a "gene machine"
• Extraction of genes

  Extraction of the gene (containing the desired nucleotide sequence) from the donor organism using restriction endonucleases
• Restriction endonucleases


  A class of enzymes found in bacteria, used as a defence mechanism by bacteria against bacteriophages (viruses that infect bacteria, also known as phages)
• How restriction endonucleases work

  Restrict a viral infection by cutting the viral genetic material into smaller pieces at specific nucleotide sequences within the molecule
• Restriction endonucleases


  Also referred to as restriction enzymes
• How restriction endonucleases cut DNA


  Separate the two strands of DNA at the specific base sequence by 'cutting' the sugar-phosphate backbone in an uneven way to give sticky ends or straight across to give blunt ends
• Sticky ends


  One strand of the cut DNA fragment gives a longer strand than the other strand
• Blunt ends


  Strands of DNA cut straight across
• Sticky ends


  • Make it easier to insert the desired gene into another organism's DNA as they can easily form hydrogen bonds with the complementary base sequences on other pieces of DNA that have been cut with the same restriction enzyme
• Creating sticky ends from blunt ends

  Nucleotides can be added to create sticky ends
• mRNA

  Messenger RNA
• Reverse transcriptase


  Enzyme that catalyses the reaction that reverses transcription
• Isolating the desired gene using mRNA and reverse transcriptase


  1. Isolate mRNA for the desired gene

  2. Combine mRNA with reverse transcriptase enzyme and nucleotides to create single-stranded complementary DNA (cDNA)

  3. Use DNA polymerase to convert single-stranded cDNA into double-stranded DNA containing the desired gene
• Reverse transcriptase


  • Sourced from retroviruses
  • Catalyses the reaction that reverses transcription
• Technique for isolating desired gene using mRNA and reverse transcriptase


  • Easier for scientists to find the gene
  • Specialised cells make very specific types of mRNA
  • mRNA (and therefore cDNA) does not contain introns
• Artificial synthesis using a "gene machine"

  1. Knowledge of the genetic code

  2. Generate the nucleotide sequence

  3. Produce short fragments of DNA

  4. Join fragments to make longer sequences

  5. Insert into vectors
• Artificial gene synthesis

  • Scientists are becoming more familiar with the base sequences for our proteins (proteome)
  • Use computers to generate the nucleotide sequence (rather than an mRNA template) to produce the gene
• Applications of artificial gene synthesis


  • Create novel genes contained in vaccines
  • Synthesise new bacteria genomes
• specificity of restriction enzymes can be investigated using extracted DNA and gel electrophoresis
• • Gel electrophoresis is a technique used widely in the analysis of DNA. During electrophoresis, an electric current is used to separate the DNA molecules according to their size / mass and their net (overall) charge
  • The separation occurs because:
  • DNA is negatively charged due to the phosphate groups and so when placed in an electric field the molecules move (migrate) towards the positive electrode
  • Different sized molecules move through the gel at different speeds. The tiny pores in the gel result in smaller molecules moving quickly, whereas larger molecules move slowly
• Restriction fragments


  DNA fragments produced by restriction enzymes
• Separation of Restriction Fragments Using Electrophoresis

  1. Extract DNA sample

  2. Digest DNA with restriction enzymes

  3. Place restriction fragments in well near negative electrode

  4. Apply electric current

  5. Fragments migrate through gel at different speeds based on size
• When a sample of extracted DNA is digested (hydrolysed) by restriction enzymes, a number of restriction fragments of different lengths are produced
• The number and size of these restriction fragments can be found using gel electrophoresis followed by visualisation of the DNA
• Visualisation of Restriction Fragments

  1. Restriction fragments move through the gel at different speeds due to their different sizes (lengths)

  2. Bands of restriction fragments are formed in the gel after electrophoresis
• DNA is colourless, so the restriction fragments must be treated in such a way so that these bands can be seen