manipulating genomes

• restriction enzymes - used for cutting dna at specific points called palindromic sequences

  • each enzyme is specific for a certain base sequence
  • dna sugar phosphate backbone is cut at the restriction site
  • a hydrolysis reaction breaks the sugar phosphate backbone
  • a staggered cut is given leaving exposed bases - sticky end
  • sometimes a blunt end remains
• PCR, contains; dna, primers, free nucleotides, taq polymerase

  1. denaturation - hydrogen bonds break between dna strands forming 2 single stranded dna molecules
  2. annealing - primers bind to strands with hydrogen bonds
  3. extension - dna polymerase adds free nucleotides in 5' to 3' direction, complementary base pairing takes place. taq polymerase starts at the primer
• pcr

  • only short sequences
  • a primer is needed to start the process
  • a cycle of heating and cooling is required
  • taq polymerase is more likely to make mistakes
• gel electrophoresis

  • dna samples are treated with restriction enzymes to cut dna into fragments
  • fragments are placed into wells at negative end of the gel
  • gel is immersed into a tank of buffer solution
  • electrodes are attached to each end of the gel and a current is passed through
  • the position of the fragments can be showing using a dye
• gene probes - dna probes are short strands of dna, they have a base sequence complementary to the section of dna

  binding by complementary base pairing = annealing

  • can be: radioactively labeled and seen on an X-ray or fluorescently labelled and seen under UV
• uses of gene probes

  • locate a specific gene for genetic engineering
  • identify the gene in a variety of genomes
  • identify the presence of a particular allele for a genetic disease
• dna profilng process

  1. extraction
  2. digestion - restriction endonuclease cut dna
  3. separation - gel electrophoresis or southern blotting
  4. hybridisation - dna probes added to label fragments
  5. development - reveals dark bands where gene probes have attached
• pharmacogenics - the study of how an individuals genome affects the body's response to drugs
• principles of dna sequencing

  • contains: taq polymerase, many copies of single stranded template dna, free nucleotides, modified termination dna with fluorescent marker, primers
• process of dna sequencing

  1. primer attached at 3' end of template strand
  2. dna polymerase attaches and complementary base pairing takes place
  3. polymerase enzyme stops when terminator nucleotide reached
  4. final nucleotide tagged with specific colour for the base
  5. mixture pulled along capillary tube by electrophoresis
  6. laser reads the colour sequence which tells order of bases
• next generation sequencing

  • high through out sequencing
  • shotgun sequencing
  • while genome sequencing
• bioinformatics - the development of the software needed to organise and analyse raw biological data, including the development of algorithms
• computational biology - using models to predict

  • the source of a disease outbreak
  • vulnerable populations to a disease
  • a specific vaccine for target antigens
• synthetic biology - date design and construction of artificial biological pathways
• genetic engineering - the genetic material of an organism can be changed. The gene form one organism can be placed into another organism for by the gene
• using biological systems in industry

  • production of drugs from microbes
  • immobilised enzymes
• synthesis of new genes - scientists have attempted to synthesise functional genes and use the to replace faulty ones
• biosensors - modified bacteria can detect chemicals in the environment
• transgenic - when the gene from one organism is placed into another
• reasons for genetic engineering

  1. improving features - resistance to weedkillers, animals promoting muscle growth
  2. allowing organisms to synthesise useful products - insert gene for human insulin into bacteria
• process of genetic engineering example spider silk from goats

  1. silk gene cut from spiders
  2. a body cell removed from the gait and enucleated
  3. the nucleus and silk gene fused with dna ligase
  4. an egg cell taken from another goat
  5. egg cell is enucleated
  6. enucleated egg cell and nucleus with silk gene fused
  7. egg stimulated to divide into embryo
  8. embryo into surrogate
  9. surrogate produces transgenic offspring
• overview of genetic engineering - 1. obtaining the gene

  • dna probe is used to locate gene
  • gene cut using restriction enzymes
  • automated polynucleotide sequencer can produce gene
  • reverse transcription of mRNA
• overview of genetic engineering - 2. gene inserted into a vector

  can use a plasmid from a bacteria

  plasmid containing desired gene cut with same restriction enzyme and dna ligase combines new plasmid with an old plasmid

  • a recombinant plasmid is created
• overview of genetic engineering -3 vector inserts gene into cells

  heat shock - ice cold calcium chloride solution, increases permeability of cell membranes

  electroporation - small electric current applied

  liposomes - dna wrapped in lipid molecules

  viral transfer - vector is a virus

  ti plasmids - plants infected via the soil
• direction method of introducing gene into recipient cell

  • gene gun - small pieces of gold are coated with the dna and shot into cells
  • microinjection - dna is injected using a very fine micropipette into host cell
• identifying transformed cells

  1. fluorescent marker genes - gene inserted into plasmid alone with desired gene, bacteria that have taken up plasmid will glow
  2. antibiotic marker genes - some plasmids contain 2 different antibiotic resistant genes, if this has worked and the bacteria takes up the plasmid, it will no longer have resistance to the antibiotic
• replica plating

  • the master plate contains standard nutrient agar and all bacterial colonies grow
  • another block is placed onto master plate and bacteria stick to it
  • block then placed onto agar that contains antibiotic
  • bacteria that have taken up plasmid with antibiotic resistant gene and desire gene will grow
• ethics of genetic manipulation

  • microbes - can be very effective, but could end up with more disease resistant strains
  • plants - can be used to increase yeild, reduced biodiversity
  • animals - gremlin cell therapy, welfare must be considered
• gene therapy - using genetic technology to treat genetic disorders, getting a functional allele into cells that contain disfunctional alleles of the gene
• somatic cell gene therapy - body cells

  adding genes (augmentation) - functioning copy of faulty gene inserted

  killing specific cells - genetic techniques can be used to make cells vulnerable to own immune system

  issues: genes into target cells difficult, short lived treatment, not in offspring
• germline gene therapy - gametes or embryos

  • cells in the embryo can specialise into any cell type
  • allele placed into these and every cell contains copy
  • issues: unethical
• problems with vectors

  • immune response - attacks foreign molecules
  • inefficient - uptake is low
  • short lived - does not always integrate with genome
  • viral vectors - virus could cause disease
  • side effects - chance of cancer if gene inserted into repressor gene