Manipulating genomes

• Purpose of PCR


  • To amplify DNA
  • To make copies of DNA
• Reason for using PCR

  • In a forensic crime scene where only a small amount of DNA is available
• Stages of PCR


  1. Heat DNA to high temperature to break hydrogen bonds (denaturation)

  2. Cool solution slightly to allow primers to anneal

  3. DNA polymerase enzyme (from extremophiles) adds free nucleotides to form new DNA strands
• DNA amount doubles with each PCR cycle
• Primer


  Short sequence of DNA bases that is complementary and specific to the part of the DNA to be replicated
• DNA polymerase used in PCR comes from extremophiles (bacteria that live in hot springs)
• The number of DNA molecules after n PCR cycles is 2^n
• Uses of DNA profiling

  • Paternity tests
  • Crime scene testing
  • Genetic screening
• Introns


  Non-coding regions of DNA
• Introns are used for DNA profiling because the non-coding DNA is most likely to be different between people, unlike the coding DNA (exons) which is similar
• DNA profiling process


  1. Extract DNA

  2. Amplify DNA using PCR

  3. Cut DNA using restriction enzymes

  4. Separate DNA using gel electrophoresis

  5. Visualize DNA using radioactive probe or fluorescent protein
• Purpose of DNA sequencing


  • To store the sequence on a database and allow quick comparisons between members of the same species or different species
  • To see if there are any links between genetic diseases
  • To predict amino acid sequences or protein structures
  • To do genetic profiling
• Steps in Sanger/chain terminator technique

  1. Extract DNA

  2. Cut DNA into fragments

  3. Amplify fragments using PCR

  4. Add DNA nucleotides, DNA polymerase, and primers to four different solutions with different terminator bases

  5. Separate DNA fragments by mass using electrophoresis

  6. Read DNA sequence
• Terminator bases


  Bases that stop DNA polymerase from adding more nucleotides, resulting in DNA fragments of different lengths
• Faster DNA sequencing techniques involve massive parallel sequencing, where many DNA sequences can be determined simultaneously