manipulating genome

• Genome


  All genetic material in an organism
• Introns


  Noncoding DNA that would be removed
• Exons


  DNA with bases that would code for proteins and would remain
• Satellite DNA


  Short sequences of DNA repeated
• Types of satellite DNA


  • Minisatellites (VNTRs) - non-coding
  • Microsatellites (STRs)
• Satellite DNA


  • Same positions on chromosomes
  • Number of repeats varies from person to person
  • Identical twins have identical pattern
  • Relatives/siblings have similar but not identical pattern
• PCR (Polymerase Chain Reaction)


  1. Sample, free nucleotides, primer DNA, DNA Taq polymerase mixed with buffer

  2. Primer anneals to complementary strand
• PCR Step 1: DNA Separation


  1. Denatures the DNA strands

  2. Hydrogen bonds between bases break
• PCR Step 2: Annealing


  DNA primer anneals to ends so replication can occur
• PCR Step 3: DNA Synthesis


  1. Optimum temp at 75C for 1 min for Taq Polymerase to work best

  2. Adds bases to primer using complementary base pairing

  3. Taq polymerase synthesises DNA and amplifies
• Differences between PCR and DNA replication


  PCR only replicates sections of DNA, uses Taq polymerase, has constantly changing heating and cooling

  DNA replication replicates the whole chromosome, uses DNA polymerase, has constant temperature due to homeostasis
• Uses of PCR


  • Genetic testing/screening
  • Studying evolutionary relationships
  • Forensic analysis of evidence
  • Genetic engineering
  • Paternity testing
• Electrophoresis


  1. Uses restriction enzymes to make DNA into smaller sizes

  2. DNA fragments in agarose gel strip wells

  3. Electric current makes DNA move to cathode

  4. Rate of movement depends on mass/length of DNA

  5. Gel resists movement, smaller pieces move faster
• Southern Blotting


  1. Gel placed in alkaline buffer to denature and separate bands

  2. Transfer to nylon sheet, placed over gel, covered in absorbent paper drawing liquid out and DNA fragments stick to nylon sheet

  3. Heat fixed using UV light

  4. DNA visualised using radioactive/fluorescent DNA probes
• Terminator bases


  Modified versions of 4 nucleotide bases that stop DNA synthesis
• Sanger Sequencing


  1. DNA mixed with primer, DNA polymerase, normal nucleotides + terminator bases

  2. Mixture in thermal cycler to rapidly change temperature at intervals

  3. Primer anneals, DNA polymerase adds free nucleotides, terminator base stops synthesis

  4. Many DNA fragments of different lengths produced

  5. DNA fragments put into capillary tube with fluorescent markers on terminator bases

  6. Lasers detect colours and order to identify regions linked with specific diseases
• Massively Parallel Sequencing


  1. Millions of DNA fragments amplified by bridge amplification PCR to make identical clusters

  2. Coloured terminator bases added to stop reaction

  3. Clusters imaged
• The Human Genome Project maps out the entire human genome using DNA sequencing
• Bioinformatics


  Development of software to organise and analyse raw biological data
• Computational Biology


  Builds theoretical models to predict what happens in different situations
• Uses of genome analysis


  • Find patterns in DNA related to human diseases
  • Identify source of infection e.g. MRSA
  • Identify antibiotic resistant bacteria to target antibiotics effectively
  • Track progress of outbreaks
  • Identify useful genome regions for drug development
• DNA Barcoding


  Identifies sections of genome common to all species (conserved) to determine species
• Evolutionary Relationships


  Determined by basic mutation rate of DNA comparison
• Proteomics


  Study of organism's entire protein complement, including amino acid sequencing
• Spliceosomes


  Pre-mRNA modified to mature mRNA, spliceosome enzymes join exons together
• Synthetic Biology


  Genetic engineering, gene therapy, artificial genomes, use of biological systems in industry
• Genetic Engineering


  The manipulation or changing the genes of an organism
• Genetic Engineering Step 1: Isolation of Desirable Gene


  Using restriction endonucleases to cut DNA at specific base sequences and expose sticky ends

  Isolating mRNA and using reverse transcriptase to produce complementary DNA
• Genetic Engineering Step 2: Vector Insertion


  Bacterial plasmid cut by same restriction endonuclease, complementary sticky ends, DNA ligase joins to form recombinant DNA
• Genetic Engineering Step 3: Transformation


  Transferring recombinant DNA to host cell using electroporation or heat shock
• Marker Genes


  Fluorescent markers like GFP show if gene inserted correctly
• Organisms used in Genetic Engineering

  • Prokaryotes - insulin, human growth hormone, clotting factors, antibiotics, vaccines

  Plants - GM bacteria causing tumours and transgenic plants

  Animals - Monoclonal antibodies, electrofusion used
• Pros and Cons of Genetic Engineering

  • Microorganisms - Producing insulin and vaccines, ethical concerns of modifying pathogens

  Plants - Pest/drought/disease resistance, nutritional value, ethical concerns of reduced biodiversity and antibiotic resistance

  Animals - Immunity, faster growth, pharming, ethical concerns of embryo destruction and animal wellbeing
• Patenting and Tech Transfer

  3rd world countries unable to afford GM seeds due to patents, companies selling seeds
• Pharming


  Creating animal models by adding/removing genes to develop certain diseases, producing human proteins
• Gene Therapy in Humans

  • Somatic cell therapy - insert healthy allele to replace mutant, temporary solution

  Germ line therapy - insert healthy allele into germ cells, cures genetic disease but potential unknown impact, illegal in humans