manipulating genomes

• DNA sequencing 


  technique to allow genes to be isolated and read to work out sequence of nucleotide bases
• Sanger technique

  1. primer anneal to 3' end
  2. when terminator bases attach the reaction stops
  3. different fragments are formed
  4. sorted by size by electrophoresis
  5. identify base by distance travelled
• automated DNA sequencing 


  same as Sanger but uses fluorescent dye instead of radioactive labels -> much faster and safer
• high throughput sequencing 


  does not involve chain termination

  e.g pyrosequencing

  • sequencing by synthesis
  • each base identified by a flash of light
• nanopore technique 


  flow of ions through nanopore creates current

  • bases moving through at same time alters current flow
  • change in current detected by computer
• uses of DNA sequencing

  whole genome sequencing -> pathogens

  enables particular strains to be studied and vaccines to be produced e.g Ebola
• bioinformatics

  development of software and computing tools to organise , store and analyse raw biological data generated
• advantages of bioinformatics 


  allows large amount of data to be stored

  format is universal

  enables access on DNA and proteins
• genome


  all the genes possessed by an organism or a population
• human genome project 


  only 2% codes for proteins -> rest is junk DNA

  99.9% is identical in all humans
• using genomes for comparison between species

  indicates genes that work well tend to be conserved by evolution e.g insulin
• using genomes for evolutionary relationships

  the more DNA that has the same sequence=the more closely related
• using genomes for variation between individuals

  share the same genes but have different alleles -> single nucleotide polymorphism (SNPs)
• using genomes to predict aa sequence 


  need to know which parts of genes code for introns and exons
• using genimes to explain role of a particular genes


  identify genes and base pairs that code for disease

  genetic screening e.g CF
• synthetic biology


  the design and construction of novel artificial biological pathways, organism or devices or the redesign of existing natural systems
• examples of synthetic biology

  genetic engineering e.g insulin

  immobilised enzymes

  synthesis of new genes to replace faulty ones e.g CF

  synthesis of an entire organism
• epidemiology 


  study of the spread of diseases in populations, enables determining most effective control method
• DNA profiling 


  producing an image of the patterns in the DNA of individuals - uses patterns of introns
• satellite DNA


  short sequences of DNA repeated many times in introns
• mini satellite or variable number tandem repeats

  sequencing of 20-50 base pairs repeated up to several hundred times

  may be highly variable
• microsatellites or short tandem repeats 


  2-4 base pairs repeated 5-15 times
• tandem repeats 


  repetitive segments of DNA not used to code for proteins
• DNA profiling procedure

  1. extraction -obtain DNA from issue samples
  2. digestion - digest DNA into small fragments using restriction enzymes
  3. separation - fragments separated by electrophoresis
  4. development - banding patter produced - use of dyes, UV light, probes
  5. compare samples
• polymerase chain reaction 


  used to rapidly produce a very large number of copies of a particular length of DNA
• PCR method

  1. denaturation-DNA heated to separates strands 95°C
  2. annealing-primer added 55-68°C
  3. elongation-taq polymerase builds up DNA 72°C
• applications of PCR

  • tissue typing
  • detection of oncogenes
  • detecting mutations
  • identifying viral infections
  • monitoring spread of disease
  • forensic science
  • research
• electrophoresis 


  used to separate DNA fragments based on their size - 1 bp difference
• electrophoresis method

  1. fragment placed in well with agarose gel
  2. placed in electrophoresis tank with buffer
  3. electrical current passed through
  4. DNA fragments carry electrical charge so travel to positive end
  5. shorter fragments travel further
• similarities between chromatography and electrophoresis

  both separate components by size
• difference between chromatography and electrophoresis

  separated by charge vs relative solubility

  solution is a buffer rather than solvent
• southern blotting 


  process to transfer DNA to a membrane where molecules can be identified
• southern blotting method

  • gel covered with nylon membrane and absorbent paper towels
  • DNA drawn up onto membrane by capillary
  • dyes/DNA probes used to stain
  • show up as black bans on x-ray film
• separating proteins

  • uses charge detergent
  • removes surface charge
  • moves according to molecular mass
• DNA probes 


  short single stranded piece of DNA (50-80 nucleotides) complimentary to DNA being investigated
• genetic engineering 


  using technology to change the genetic material of an organism
• recombinant DNA

  DNA that has been altered by genetic engineering , joining together pieces of DNA from different species
• transgenic organism 


  organism to which the new gene has been added
• restriction enzymes 


  endonuclease - cut DNA at specific recognition sites
• electroporation 


  used to introduce a vector - uses pulse of electricity to increase membrane permeability