manipulating genomes

• DNA sequencing - identifying the base sequence of a DNA fragment
• How has sequencing methods changed over time?

  • Used to be manual, now automated
  • entire genome can now be read
• • knowing the sequence of a gene allows us to predict the sequence of amino acids that will make up the polypeptide it produces
  • allows for development of synthetic biology
• knowing the sequence of a gene allows us to predict the sequence of amino acids that will make up the polypeptide it produes

  this allows for development of synthetic biology
• DNA profiling - identifying the unique areas of a persons DNA in order to create a profile that is individual to them
• Polymerase chain reaction (PCR) makes millions of a copies of a fragment which are then cut at different lengths in order to be sequenced
• genome - all genetic information an organism contains
• exons - genes that code for proteins only make up 2% of total DNA
• satelite DNA discovered with introns, telomeres and centromeres have short sequences of DNA repeated many times
• minisatellites - sequence of 20-50 base pairs that can repeat 50+ times (variable number tandem repeats, VNTRs)
• microsatellites - smaller sections of 2-4 base pairs, repeated 5 to 15 times (short tandem repeats, STRs)
• producing a DNA profile

  1. DNA extraction
  2. digesting the sample
  3. DNA fragment seperation
  4. hybridisation
  5. analysis
• DNA profiling uses

  • forensic science
  • paternity testing
  • immigration cases
  • phylogeny
  • genetic testing
• DNA extraction

  • taken from tissue sample (WBC)
  • PCR enables us to work with small samples

  PCR works by

  • amplifying DNA using DNA polymerase
  • Primers start copying DNA
  • DNA replication
• DNA extraction

  1. 95 degrees for 30 secs denatured DNA by breaking H bonds
  2. 55 degrees allows primers to attach
  3. 72 degrees optimum temperature for DNA polymerase to work
• Sample digestion

  • restriction endonucleases used to cut DNA into smaller fragments
  • different endonucleases cut DNA at specific sites known as restriction/recognition sites
• Fragment seperation

  • DNA fragments under gel electrophoresis
  • DNA fragments put into wells in agarose gel strip
  • Electrical current passed through electrophoresis plate
  • Gel placed into alkaline bugger solution to denature DNA fragments which exposes bases between 2 DNA strands
  • Strands are transferred to nylon membrane by southern blotting, fixed in place by exposure to UV light
• Electrophoresis

  • DNA fragments move through gel from cathode to anode due to phosphate group being negatively charged
  • rate of movement depends on mass/length of DNA fragment
• Hybridisation

  • Radioactive/florescent DNA probes are added in excess onto the membrane
  • Bind to complementary DNA under particular conditions
  • Excess probes are washed off
• analysis

  • radioactive labels added to DNA probes the membrane then undergoes an x-ray to form image of the DNA pattern
  • If fluroscent labels added to membrane then its placed under UV ight so tags glow to show DNA pattern
  • Bar pattern shown will be unique to every individual except identical twins
• terminator bases give fluroscent tags

  • a = green
  • g = yellow
  • t = red
  • c = blue
• Sequencing process involves DNA mixed with primers, DNA polymerase, excess of normal nucleotide and termination bases placed in thermal cycler.
• At 96 degrees, DNA strand separates.
• At 50 degrees, primers anneal to DNA strand.
• At 60 degrees, DNA polymerase starts to build up new DNA strands.
• When a terminator is used instead of a nucleotide, no more bases can be added, causing many bases to vary in length.
• DNA fragments are separated or capillary sequencing, similar to gel electrophoresis, out of capillary tubes.
• Fluroscent tags are used to identify terminator bases using lasers.
• The order of nucleotides shows the sequence of the new complementary strand.
• Computer analyses overlaps in code in fragments.
• next generation sequencing

  • sequencing occurs on a slide called flow cell allowing millions of DNA fragments to be replicated in-situ in PCR
  • known as massively parallel sequencing
  • being constantly refined and integrated with state-of-the-art-computers
  • high throughput methods are extremely fast and efficient sequencing bacterium genomes in less than 24 hours
• bioinformatics

  • computer software anf tools developed to organise/analyse raw biological data
  • includes: algorithms, statistical tests, mathematical models
• computational bio

  • constructs theoretical models of biological systems
  • enables different scenarios to be tested and predictions to be formulated
• Genomics

  • applies DNA sequencing and computational bio and analyse the structure and function of genomes
  • enables: analysis of human genimes, analysis of pathogen genomes, species identification (DNA barcoding), revision of evolutionary trees
• Proteomics

  • study and sequencing of amino acids that form an organisms entire protein collection
  • discovered that the protein collection observed within an organism didnt always match the predicted outcomes based upon the genome sequence
  • result of spliceosomes and protein modification
• Synthetic bio

  • emerging areas of research aimed at artifically replicating biological processes and systems
  • includes techniques e.g. genetic engineering, exploiting natural processes in an industrial context
  • replacement of faulty genes by synthesising new versions
  • artificial synthesis of new organisms