Manipulating genomes

• Outline the Sanger/chain termination technique

  • extract the DNA and cut into fragments of various lengths and amplify it
  • sequence the DNA by adding it to 4 different solutions
  • DNA is split into single strands, and copied multiple times
  • DNA polymerase adds nucleotides to the template to start rebuilding new DNA strands
  • When a terminator base is added DNA synthesis stops and tagged with a fluorescent colour
  • This produces DNA fragments
• How are DNA fragments separated by length?

  • gel electrophoresis
• What is gel electrophoresis?

  • separates DNA at different lengths
  • DNA fragments are placed in the wells at the top of the agar gel (using a pipette)
  • an electric current is applied over it
  • DNA moves towards the positive electrode at different rates (small fragments are quicker)
• Examples of sequencing techniques that are faster than Sangers

  • high throughput sequencing
  • next generation sequencing
  • whole genome sequencing
• What is massive parallel sequencing

  • you can sequence many DNA at the same time
• Why is DNA attracted to the positive electrode?

  • DNA is negatively charged
• Shorter DNA fragments will be closer to the anode in electrophoresis because it has less resistance as it passes through the gel
• Why is it important during electrophoresis that it is run for a set amount of time?

  • eventually all the DNA will end up at the anode
• What are the two ways to see DNA after you have your reading from your electrophoresis?

  1. using radioactive DNA probes and X rays to see the DNA
  2. using green fluorescent protein DNA probe and UV light
• What is present in the solution in the sanger technique?

  • DNA nucleotides
  • DNA polymerase
  • Primers
  • A terminator base
• Suggest why a fragment has to be fragmented before sequencing?

  • too large to sequence
  • smaller fragments will produce fewer errors and is more accurate
• What is the role of restriction enzymes?

  • cut the genome into smaller fragments
  • cut vectors
• Why is it easier to sequence a smaller genome?

  • fewer lab hours spent
  • quicker
  • cheaper
  • fewer staff needed
• Outline the process of sanger sequencing

  1. extract DNA; cut into fragments of various lengths
  2. Amplify using PCR
  3. Place them into the four solutions
  4. Terminator bases stop more nucleotides adding on
  5. Add them to electrophoresis plate
  6. Information is stored to allow quick comparisons
• What is present in the four solutions in sanger sequencing?

  • DNA fragments
  • DNA polymerase
  • primers
  • a terminator base (each solution contains a different terminator base)
• Outline the process of electrophoresis

  1. pipette the four solutions into different wells
  2. pass a current through the electrophoresis plate
  3. DNA fragments move through the gel to the positive electrode
  4. DNA fragments are separated due to mass
  5. Smaller fragments have less resistance with the gel so end up closer to the bottom
• How can you view DNA?

  • southern blotting - radioactive DNA probes or x-rays can be used
  • using green fluorescent protein DNA probes and UV light
• What is throughput sequencing and next generation sequencing?

  • allow for rapid sequencing of DNA
  • multiple sets of DNA are sequenced together
  • both use massive parallel sequencing
• What is the purpose of PCR?

  • amplifies DNA
  • this is so that several copies of DNA can be made
  • the original piece of DNA is not destroyed
• What are the stages of PCR?

  • denaturation
  • annealing
  • synthesis
• Outline denaturation in PCR

  • heat the DNA
  • molecules gain kinetic energy and hydrogen bonds break
  • 95 degrees celcius
• Outline annealing in PCR

  • primers are complementary to bases
  • primers recognise target DNA to be amplified
  • 55 degrees Celsius
• Outline synthesis in PCR

  • free nucleotides pair up with exposed bases by complementary base pairing
  • tac polymerase is used to join the sugar phosphate backbone
  • 72 degrees celcius
• What is a thermal cycler?

  • the machine used in PCR
  • it controls and changes the temperature at programmed timings
• How do restriction endonucleases work in genetic engineering?

  1. it is used to cut the desired gene from DNA
  2. creates sticky ends which makes it easier to insert the desired gene into the vector
  3. The vector is cut using the same restriction enzyme to produce complementary sticky ends
  4. the desired gene is inserted into the vector at a marker gene and joined by DNA ligase
• What are the ways in which recombinant DNA can be inserted into the host cell?

  • it can be mixed in a Calcium solution and heated to increase kinetic energy of the phospholipids to increase membrane permeability
  • electroporation - an electric shock which makes pores in the membrane
  • electrofusion
• BIOINFORMATICS:

  • allows access to large amounts of data on DNA and proteins
  • information is universal
  • allows rapid comparison of sequences with newly sequenced alleles
  • amino acid sequence/protein structures held in database
  • computer modelling of new protein structure from base sequencing