manipulating genomes

• Manipulating genomes

  Includes DNA sequencing techniques and sequencing projects to read genomes of organisms for various applications like identifying potential medical problems
• The human genome consists of three billion base pairs and approximately 20 to 23,000 genes
• The proteome is all the proteins that a cell can produce
• Sequencing methods are continuously improved, updated, and automated to increase speed and accuracy
• DNA sequencing

  Techniques involve Terminator bases to stop DNA synthesis, creating new strands of DNA with fluorescently labeled bases for identification
• The principles behind DNA sequencing have become faster and automated, using Terminator bases to terminate DNA synthesis at different points
• DNA sequencing

  1. Terminator bases are labeled with different fluorescent colors to identify them

  2. DNA fragments are separated using gel electrophoresis according to their length

  3. Fragments are arranged by length and Terminator base fluorescence indicates the base

  4. High throughput sequencing processes many fragments simultaneously for efficiency

  5. Genome sequencing allows comparisons within and between species

  6. Used for analyzing pathogens genomes and identifying sources of infection, antibiotic-resistant bacteria, tracking pathogen spread, monitoring epidemics and pandemics, identifying regions for new drugs, improving species classification, and understanding evolutionary relationships

  7. Predicting all proteins an organism can produce from its genome is complex due to varying gene predictions and the complex relationship between genotype and phenotype
• Synthetic biology

  1. Enabled by sequencing genomes, involves creating artificial pathways, organisms, devices, or redesigning natural systems

  2. Examples include genetic engineering, use of biological systems in industry, creating new genes, and organisms
• 95% of human DNA is made up of introns, which are non-coding DNA sequences within genes consisting of variable number tandem repeats (VNTRs)
• VNTRs analysis through genetic fingerprinting determines relatedness based on the similarity of VNTRs
• Genetic fingerprinting

  Collect sample, extract DNA, amplify DNA using PCR, digest DNA with restriction endonucleases, separate DNA fragments using gel electrophoresis, add alkaline to split DNA into single strands, hybridize single strands with DNA probes, rinse gel, and analyze using probes with radioactive or fluorescent labels
• Hydrogen bonds with those sequences


  Needed to see where those bands are because DNA itself isn't actually visible
• DNA probes binding

  1. Probes have a radioactive label or fluorescent label on them

  2. After allowing time for the probes to bind, rinse the gel

  3. Transfer all from the gel onto a nylon sheet to visualize results properly

  4. Expose the nylon sheet to x-rays to visualize radioactive labels on a gene probe or use UV light to visualize positions
• Back in 2015, at Imperial College on a school trip, bacterial DNA samples were analyzed using gel electrophoresis
• Unknown bacterial species identified as bacterial species three based on band positions in gel electrophoresis
• PCR (Polymerase Chain Reaction)


  Used at the start of Gene Technologies to create a large sample of DNA for multiple repeats and experiments
• Equipment list for PCR

  • Thermocycler machine
  • DNA fragment to amplify
  • Enzyme DNA polymerase
  • Primers
  • DNA nucleotides
• PCR


  Automated and rapid process, doesn't require living cells like in Vivo cloning
• Taq polymerase


  Special type of DNA polymerase from bacteria that naturally grow in Hot Springs, doesn't denature at high temperatures
• PCR method

  Temperature increases to 95°C to break hydrogen bonds, primer aligns opposite complementary bases, DNA polymerase attaches and adds nucleotides, temperature drops to 55°C for hydrogen bonds to form, temperature increases to 72°C for synthesis stage
• Advantages of PCR: automated, rapid, doesn't require living cells like in Vivo cloning
• In Vivo cloning is a type of cloning DNA in living cells
• Recombinant DNA Technologies involve genetic fingerprinting, gel electrophoresis, PCR, and in Vivo cloning
• In Vivo cloning

  Inserting DNA fragment into a vector (plasmid) to carry it into the host organism for transcription
• Inserting DNA fragment into a plasmid

  Using restriction endonucleases to cut DNA fragment and plasmid, adding promoter region at the start and terminator region at the end of the fragment, joining plasmid and fragment using DNA ligase at sticky ends
• Gene fragment and the open plasmid are mixed together

  Enzyme DNA ligase is added to join the plasmid and the fragment at the sticky ends, forming phosphodiester bonds between the nucleotides to create a recombinant plasmid
• Transformation

  Getting the Vector into the host cell by making the cell membrane more permeable through mixing with calcium ions and undergoing heat shock
• Heat shock


  Enables the vector to enter the host cell
• Not all bacteria get transformed, some take up plasmids without the gene of interest
• Identifying transformed cells

  Using Gene markers, checking for antibiotic resistant genes within plasmids
• Plasmids contain marker genes, often antibiotic resistant genes
• Recombinant plasmids disrupt antibiotic resistant genes to identify transformed cells
• Identifying transformed cells using antibiotic resistant marker genes

  Growing bacteria on agar plates, imprinting colonies onto agar with antibiotics, observing growth to identify transformed cells
• Genetic engineering in crops involves adding genes for pest resistance, disease resistance, and herbicide resistance to increase yields
• Genetically engineered soy plants produce BT toxin, reducing the need for pesticides and increasing yields
• Genetic engineering in crops can also manipulate DNA for longer shelf life, reduced food waste, increased nutritional value, and medicine production
• Concerns about genetic engineering in crops include gene spread to other plants, potential allergies, and expensive patented technology
• Genetic engineering in animals involves injecting modified viruses and gold-covered DNA to carry new genes into their DNA
• Genetic engineering in animals has been used to create swine flu-resistant pigs and faster-growing salmon
• Genetic engineering in microorganisms is widely used in farming