W3:Introduction to DNA

• DNA


  The primary genetic material in prokaryotic and eukaryotic cells
• Gene expression


  1. Transcription

  2. Translation
• Only a small percentage of DNA in a cell is being expressed at any given moment
• Central Dogma of Biology

  Information flow: Proteins are produced when genes on a DNA molecule are transcribed into mRNA, and mRNA is translated into proteins

  
• DNA (or RNA)


  Polymer of a sequence of nucleotides
• Nucleotide


  Chemical unit consisting of a sugar molecule, a phosphate and a nitrogenous base (Adenine, Thymine, Cytosine, Guanine)
• DNA structure


  • Nitrogenous bases from each strand bond to each other in the centre through hydrogen bonds
  • 2 hydrogen bonds formed between Adenine and Thymine
  • 3 hydrogen bonds formed between Cytosine and Guanine
  • A and T; G and C are paired on opposite strands, so the amount of A=T and G=C

  
• DNA structure


  • DNA forms a double helix
  • DNA strands run anti-parallel (opposite directions)
  • Space-filling model to illustrate the regularly spaced grooves available for binding and interaction with other cellular molecules (transcription factors etc)

  
• DNA as genetic material


  • Stores genetic information
  • Copies itself and faithfully transmits to next generation
  • Evolves to new versions
  • Expresses the genetic information

  
• DNA Replication


  1. DNA replication is semi-conservative

  2. The two strands are separated and each strand is used as a template to make a new copy

  3. Each new DNA molecule contains one new (daughter) strand and one of the original (parent) strands

  
• Similarities in DNA molecules across organisms

  • DNA molecules form a double helix
  • All DNA molecules are composed of four nucleotides (A, C, G, T)
  • DNA strands run anti-parallel
  • A and T; G and C are paired on opposite strands (amount of A=T; amount of G=C)
  • DNA undergoes semi-conservative replication

  
• Variations in DNA molecules across organisms

  • Number of DNA strands in cells (no of chromosomes)
  • Length in base pairs in chromosomes (the length of the DNA strands)
  • Number and types of genes and non-coding regions
  • The conformation of DNA: linear in eukaryotic cells but circular in prokaryotic cells

  
• Prokaryotic DNA


  • Prokaryotic cells do not have a defined nucleus
  • Prokaryotic chromosomes comprise one circular DNA molecule
  • Circular DNA is usually supercoiled; more compact
  • Most of prokaryotic DNA is coding; minimal amount of non-coding DNA

  
• Prokaryotic DNA: Operon


  The sequence of bases coding for one or more polypeptides, together with the promoter/operator and a terminator region controlling the expression of the structural gene
• Prokaryotic DNA: Plasmids


  • Small circular DNA molecules that can be transferred between bacteria
  • Contain a small number of useful genes
  • May give cells a selective advantage (antibiotic resistance, herbicide resistance)
  • Plasmids can be used to introduce new genes into prokaryotic cells
  • Recombinant plasmids, containing new gene segments, are easily constructed in the lab

  
• Bacterial cell culture


  1. Bacteria can be grown in liquid media (broth culture) or on solid media (agar plates)

  2. All broth, media, glass and plastic ware must be sterilized before use, usually by filter sterilization or autoclaving

  3. Autoclaving: high temperature and pressure (121°C, 15 psi) effectively destroys bacterial and fungal cells but may not destroy viruses or spores

  4. Cells to be cultured are introduced using aseptic techniques
• Eukaryotic DNA


  • In eukaryotic cells, DNA is organized into large linear chromosomes
  • DNA is tightly condensed, coiled and folded around each other and associated with histone proteins - nucleosomes
  • Not all DNA in a cell is accessible to regulatory proteins
  • May affect gene expression (regulation)

  
• Eukaryotic gene


  • Eukaryotic genes have a promoter to which RNA polymerase (which makes mRNA) binds, but they do not have an operator region
  • Gene expression is regulated by interactions with enhancer and silencer sequences and proteins
  • In eukaryotes, enhancers and silencers regulate gene expression from a distance
  • Non-coding DNA is also present
  • Genes may be interrupted by non-coding segments (introns) that must be edited from mRNA precursors
  • Eukaryotic mRNA have 5' cap and 3' Poly (A) tail

  
• Mammalian cell culture


  • Growing mammalian cells in culture is more challenging than growing bacterial cells
  • They require complex media, nutrients and a number of co-factors
  • Most cells also require surface attachment
  • Grown in shallow broth cultures, and incubated at 5% CO2 and 37ºC, reflecting the environment of internal cells in mammalian systems
• Viruses


  • Cellular parasites that reproduce only within living host cells
  • Genome may be one single type of DNA or RNA, protected by a protein coat and sometimes enveloped by lipids, proteins and carbohydrates
  • Classified according to the type of cell they attack: bacterial (bacteriophages), plant, animal
• Viruses as vectors


  • Introduce new genes, creating a recombinant viral genome for gene delivery
  • Has potential applications in gene therapy in the form of recombinant virus technology
• Genetic Engineering methods


  1. Identification of the molecule(s) to be produced

  2. Isolation of the appropriate DNA sequence

  3. Manipulation of the genetic code to ensure gene expression in new production host

  4. Harvesting of the product, testing and marketing

  
• Recombinant DNA (rDNA) Technology

  Methods to create new DNA molecules by isolating product gene sequence, modifying sequence for expression in production host, preparing vector DNA to accept gene sequence, and ligating gene into vector

  
• Site-Specific Mutagenesis


  • Process of including changes (mutagenesis) in certain sections (site-specific) of a particular DNA sequence
  • Mutations can be induced by chemical treatment, UV light irradiation, or viral infection
  • Genetic engineering techniques permit specific changes to be introduced into DNA with predictable outcomes

  
• Gene Therapy


  Process of correcting faulty genes that cause genetic diseases by supplying a functional gene copy or knocking out harmful/toxic proteins
• Agarose Gel Electrophoresis


  1. Separates molecules of interest based on size, shape, and charge

  2. Powdered agarose is dissolved in buffer to create a molecular sieve

  3. Negatively charged nucleic acids move towards the positive electrode, with smaller/more compact molecules moving faster
• Gel Stains


  • SYBRgreen
  • Coomasie Blue
  • Ethidium Bromide
• Mammalian cell culture


  The process of growing mammalian cells in a controlled environment outside of a living organism
• Complex media


  The liquid that mammalian cells are grown in, which contains a variety of nutrients and co-factors
• Co-factors


  Substances that help enzymes in the cells to function properly
• Surface attachment


  The requirement for most mammalian cells to be grown on a solid surface
• Shallow broth cultures


  The shallow layer of liquid in which mammalian cells are grown
• 5% CO2 and 37ºC


  The environment in which mammalian cells are incubated, reflecting the conditions inside the body of a mammal
• Mammalian cells vs bacterial cells in culture

  Mammalian cells are more complex and require a more complex environment to grow, while bacterial cells are simpler and can be grown in a basic liquid medium
• Method to sterilise cell culture and apparatus

  Filter sterilisation and autoclaving