7.1

• Polymerase Chain Reaction (PCR)

  1. Reaction mixture set up with DNA sample, primers, free nucleotides and heat-stable DNA polymerase

  2. Mixture heated to 95 degrees Celsius to separate DNA strands

  3. Mixture cooled to 50-65 degrees for primers to bind (anneal)

  4. Temperature increased to 70 degrees for DNA polymerase to create copies

  5. Cycle repeated around 30 times to amplify DNA
• DNA sequencing

  Used to predict amino acid sequence of proteins and determine links to genetically determined conditions
• DNA sequencing process

  1. DNA sample divided into 4 reactions with standard nucleotides, DNA polymerase, primers and fluorescently labelled terminator nucleotides

  2. Terminator nucleotides incorporated, stopping replication

  3. DNA fragments of different lengths produced

  4. Fragments separated by size using gel electrophoresis

  5. Fragments visualised under UV light to read base sequence
• DNA profiling process

  1. DNA fragments cut with restriction endonuclease enzymes

  2. Fragments separated and visualised using gel electrophoresis

  3. Southern blot used to transfer fragments to nylon filter and hybridise with gene probes

  4. Number of satellite repeats compared to determine relatedness
• What is a genome?

  A genome is all of an organisms DNA, including mitochondrial/chloroplast DNA
• Polymerase chain reaction (PCR) is used to amplify the DNA by making millions of copies of a given DNA sample
• Reaction mixture


  Contains DNA sample, primers, free nucleotides and heat-stable DNA polymerase
• DNA polymerase


  Enzyme involved in creating new DNA strands, also known as taq polymerase. Optimal function at 72°C
• PCR (Polymerase Chain Reaction)


  1. Reaction mixture set up
  2. Mixture heated to 95°C to separate DNA strands
  3. Mixture cooled to 50-65°C for primers to bind
  4. Temperature increased to 70°C for DNA polymerase to create copy
  5. This cycle is repeated around 30 times
• Explain the first stage of PCR

  A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and heat-stable DNA polymerase, which is an enzyme involved in creating new DNA strands
• Explain the second stage of PCR

  The mixtures and heat it is 95°C to break the hydrogen bonds between the complimentary bases and to separate the two strands
• Explain the third stage of PCR

  The mixture then caught a temperature between 50-65°C depending on the type of primers used, so the primers can bind to the strands (annealing)
• Explain the fourth stage of PCR

  Temperature is increased to about 70°, as this is the temperature DNA polymerase works at. DNA polymerise create a copy of the sample by complementary base pairing using the free nucleotides
• Explain the fifth stage of PCR

  This cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to create a DNA profile
• DNA profiling is a frantic technique used to identify criminals and test paternity
• DNA profiling consists of 4 stages:

  • Fragments of DNA are cut with restriction endonuclease enzymes
  • These fragments are separated and visualised using gel electrophoresis
  • Southern Blot - alkaline buffer solution is added, nylon filter – dry up absorbent material draw solution containing DNA fragments to the filter – fragments are visible as ‘blots’. Gene probes are added and bind with DNA (hybridisation)
  • ‘Blots’ compared and the number of satellites visualised as a graph