Manipulating genomes

• Genome


  All of the genes possessed by an individual or an organism, including the DNA in the nucleus and mitochondria
• Only 0.1% of the human genome is different, 99.9% is exactly the same across all human beings
• Types of genes in the genome

  • Exons
  • Introns
• Exons take up about 2% of the entire genome and code for proteins
• Introns were previously called 'junk DNA' but may be involved in gene expression regulation
• Satellite DNA


  Short repeated DNA sequences, including mini-satellites (variable number tandem repeats) and microsatellites (short tandem repeats)
• Mini-satellites


  • Sequence of 20-15 base pairs that repeat 50-100 times
• Microsatellites


  • Sequence of 2-4 bases that repeat 5-15 times
• Satellite DNA appears in the same area on chromosomes, but different people have different numbers of repeats
• Identical twins share the same satellite DNA patterns, while closer relatives have very similar patterns
• DNA profiling


  Identifying an individual and determining familial relationships by analysing differences in satellite DNA patterns
• DNA profiling process


  1. Extract DNA from sample

  2. Amplify DNA using PCR

  3. Cut DNA into fragments using restriction enzymes

  4. Separate DNA fragments using gel electrophoresis

  5. Transfer DNA to nylon membrane (Southern blotting)

  6. Detect DNA fragments using fluorescent or radioactive probes
• DNA profiling is commonly used in forensics to compare crime scene evidence to suspect samples
• DNA profiling can also be used for paternity testing, as the child will have a similar satellite DNA pattern to the biological father
• Polymerase Chain Reaction (PCR)


  1. Denaturation

  2. Annealing

  3. Synthesis/Amplification
• PCR


  Artificial DNA replication process to amplify DNA fragments for processing and study
• Situations where PCR is used


  • Forensics - extracting DNA from hair or skin tissue to generate a DNA profile
• There is not enough DNA in samples for study, so PCR is used to increase the concentration
• Thermal cycler machine


  • Changes temperature at set times to allow PCR steps to occur in a cycle
• Denaturation


  At 95°C, heat breaks hydrogen bonds between complementary bases, separating DNA strands
• Annealing


  Temperature decreased to 55°C, primers bind to start of target gene sequence
• Synthesis/Amplification


  Temperature increased to 72°C, DNA polymerase (from bacteria) adds complementary nucleotides, forming new DNA strands
• Human DNA polymerase would denature at 72°C, so bacterial Taq polymerase is used instead
• The new DNA strands then repeat the PCR cycle, exponentially amplifying the target sequence
• The primer design is important, with one primer binding to each DNA strand in the correct 5' to 3' orientation
• DNA sequencing


  Developed by Frederick Sanger, also called Sanger method or Sanger sequencing
• DNA sequencing was a very important breakthrough in the human genome project and actually sped up the entire project by a couple of years and saving a lot a lot of resources and money and it also actually pays way for future improvements which led to the next generation sequencing
• Ingredients for DNA sequencing

  • DNA sample
  • Free nucleotides in excess
  • Fluorescently labeled terminal bases
  • DNA polymerase
  • Primer
• DNA sequencing is very similar to PCR (polymerase chain reaction), it uses the same machine (thermal cycler) and similar ingredients
• DNA sequencing process


  1. Heat DNA sample to 95-96°C to denature

  2. Cool to 50°C for primers to anneal

  3. Heat to 60°C for DNA polymerase to add complementary bases

  4. Terminated bases stop further extension
• Repeated cycles result in fragments of different lengths
• Reading DNA sequencing data

  1. Gel electrophoresis and Southern blotting, read order of fragments from top to bottom

  2. Capillary electrophoresis with laser, read computer signal graph
• The final step is to build the original sequence from the data