Bioninja Topic 3.5

• A gene determines a particular trait by encoding for a specific polypeptide in a given organism
• Gene transfer to bacteria using plasmids makes use of restriction endonucleases and DNA ligase
• Gene modification process

  1. Isolation of gene and vector (by PCR)

  2. Digestion of gene and vector (by restriction endonuclease)

  3. Ligation of gene and vector (by DNA ligase)

  4. Selection and expression of transgenic construct
• The transfer of genes between species is called gene modification, and the new organism created is called a transgenic
• Gene transfer process

  1. Isolating gene and vector DNA

  2. Digestion of gene and vector

  3. Ligation of gene and vector

  4. Selection and expression of transgenic construct
• Because the genetic code is (almost) universal, an organism can potentially express a new trait if the appropriate gene is introduced into its genome
• DNA can be isolated from cells by centrifugation, whereby heavier components such as nuclei are separated
• Vector


  A DNA molecule used as a vehicle to carry the gene of interest into a foreign cell
• Digestion with Restriction Enzymes

  1. Incorporation of a gene of interest into a vector by cutting both with restriction enzymes at specific recognition sites

  2. Cleavage of the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)

  3. Double digestion with two different 'sticky end' restriction endonucleases to ensure correct orientation of gene insertion and prevent vector re-annealing without the desired insert
• Selection and Expression

  1. Introduction of the recombinant construct into an appropriate host cell or organism

  2. Achievement through transfection (for eukaryotes) or transformation (for prokaryotes)

  3. Common use of antibiotic selection to identify cells with successfully incorporated recombinant construct

  4. Growth of transgenic cells in the presence of antibiotic due to the plasmid vector containing an antibiotic resistance gene

  5. Expression of the desired trait encoded by the gene of interest in isolated and purified transgenic cells
• Isolation of transgenic construct

  1. Step 1: Isolating gene and vector DNA from cells by centrifugation

  2. Specific amplification of the gene of interest via polymerase chain reaction (PCR)

  3. Generation of gene sequences from mRNA using reverse transcriptase to obtain cDNA

  4. Usage of a vector as a DNA molecule to carry the gene of interest into a foreign cell, commonly bacterial plasmids

  5. Modification of plasmids for further functionality such as selection markers, reporter genes, and inducible expression promoters

  6. Other types of vectors include modified viruses and artificial chromosomes
• Ligation of Vector and Insert

  1. Insertion of the gene of interest into a plasmid vector cut with the same restriction endonucleases

  2. Splicing of gene and vector together via complementary base pairing of sticky ends

  3. Formation of a recombinant construct by DNA ligase joining the vector and gene with a covalent phosphodiester bond
• Common Features of a Typical Plasmid Vector

  • Step 2: Digestion with Restriction Enzymes
  • Step 3: Ligation of Vector and Insert
  • Step 4: Selection and Expression
• Sticky End vs Blunt End Restriction Enzymes


  Explanation of the difference between 'sticky end' and 'blunt end' restriction enzymes
• Bacterial plasmids


  Commonly used as vectors due to their capability of autonomous self-replication and expression
• PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps: Denaturation, Annealing, Elongation
• Stages of PCR

  1. Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min)

  2. Annealing – DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)

  3. Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min)
• PCR

  1. PCR can be used to amplify small amounts of DNA

  2. PCR is an artificial method of replicating DNA under laboratory conditions

  3. PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample

  4. Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (2^30)
• Once large quantities of DNA have been created, other laboratory techniques are used to isolate and manipulate the sequences
• Polyacrylamide Gel Electrophoresis (Proteins)

  1. Proteins must be treated with an anionic detergent (SDS) to linearise and impart a uniform negative charge

  2. Protein samples are placed into a polyacrylamide gel and sizes are compared against known industry standards

  3. Separated proteins are identified by staining with specific monoclonal antibodies (Western blotting)
• Gel electrophoresis is a laboratory technique used to separate and isolate proteins or DNA fragments based on mass/size
• DNA is negatively charged due to the presence of a phosphate group on each nucleotide
• Gel electrophoresis

  1. Used to separate proteins or fragments of DNA according to size

  2. Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel

  3. Smaller samples move faster through the gel matrix, causing samples of different sizes to separate as they travel at different speeds
• Proteins may be folded into a variety of shapes affecting size and have positive and negative regions
• Agarose Gel Electrophoresis (DNA)

  1. DNA samples are placed into an agarose gel and fragment size is calculated by comparing against known industry standards

  2. Specific sequences can be identified by incorporating a complementary radiolabelled hybridisation probe, transferring the separated sequences to a membrane, and visualising via autoradiography (Southern blotting)
• Overview of Gel Electrophoresis

  • Both DNA and proteins are separated based on mass/size
  • DNA may be cut into fragments using restriction endonuclease
  • DNA samples are placed into an agarose gel and fragment size is calculated by comparing against known industry standards
  • Specific sequences can be identified by incorporating a complementary radiolabelled hybridisation probe and visualising via autoradiography (Southern blotting)
  • Proteins must be treated with an anionic detergent (SDS) to linearise and impart a uniform negative charge
  • Protein samples are placed into a polyacrylamide gel and sizes are compared against known industry standards
  • Separated proteins are identified by staining with specific monoclonal antibodies (Western blotting)