BIO 220

• Polymerase Chain Reaction (PCR)


  A way to make many copies (millions!) of a DNA sequence
• PCR


  • Uses Taq DNA polymerase to replicate DNA in vitro
  • Requires knowledge of the sequence of interest
  • Relies on two primers that match sequences on both sides/ends of the gene
• Requirements of PCR


  1. The reaction mix contains dNTPs, a DNA template, forward and reverse primers, and Taq polymerase

  2. PCR cycles through three steps repeatedly: Denaturation, Annealing, Extension

  3. Repeating these steps just 20 times can yield over a million copies from a tiny amount of template DNA
• PCR Components


  • Template DNA
  • Primers (forward and reverse)
  • dNTPs
  • Taq polymerase
• Applications of PCR


  • Forensic scientists can amplify DNA from tiny blood or hair samples to determine paternity or identify suspects
  • Evolutionary biologists investigate DNA to determine ancestral relationships
  • Genetic counselors identify disease alleles in parents
  • Physicians identify bacterial and viral pathogens and diagnose subtypes of cancers
• DNA Sequencing


  Determining the sequence of bases in genes
• The amino acid sequence of the protein can provide clues to the protein's function
• Evolutionary relationships can be inferred by comparing sequences of the same gene in different species
• Sanger sequencing


  Developed dideoxy sequencing/Sanger sequencing
• Next-generation sequencing


  Much more rapid, based on amplifying DNA, can sequence millions of DNAs in a single run, produces short sequence reads of 50–200 bases, must be overlapped to produce a complete genome, can be compared to a "reference genome" to very quickly identify differences in an individual
• Shotgun sequencing


  Involves sequencing many copies of a genome in short fragments, fragments overlap in many regions, overlap regions are used to assemble the genome, the speed and accuracy of genome sequencing is accelerating while the cost is decreasing
• Bioinformatics


  Fuses biology, computer science, and mathematics, manage and analyze sequence data, vast searchable databases of sequence information, researchers can compare newly discovered genes to previously studied genes
• Cells


  • Must be responsive to changes in environmental conditions
  • In multicellular organisms must communicate and cooperate with each other
  • Must respond to stimuli/information present in the environment (and sent by other cells)
  • Must physically interact with other cells
  • In a tissue or organ must coordinate function
• Extracellular Matrix (ECM)


  • Fiber composite secreted by most animal cells
  • Fibrous component is mostly collagen, a cable-like protein that binds together to form fibrils or fibers
  • Ground substance is made of proteoglycans, proteins attached to many polysaccharides that attract water and form a gel
• Collagen protein


  • Consists of three polypeptide chains that wind around one another to form the fibrous component of the animal ECM
• Proteoglycan


  • Complexes of gelatinous proteoglycans form the ground substance of the animal ECM
• Tissues


  • Composed of similar cells that function as a unit
  • The composition and amount of ECM varies in tissues
• ECM


  Provides cells with physical and biochemical cues/information that direct cell fate decisions and enable cells to function properly
• Integrins


  • Family of membrane proteins found only in animals
  • Provide connection between ECM and cytoskeleton
  • Bind to ECM molecules outside cell
  • Interact with a wide array of proteins inside cell
• Multicellularity


  The basis is physical connections between cells
• Tight junctions


  • Seal cells together with a watertight seal
  • Usually found in epithelia–tissues that line surfaces
• Desmosomes


  • Connect the cytoskeletons of adjacent cells
  • Resist pulling and shearing forces
  • Common in epithelial and muscle tissue
• Gap junctions


  • Direct connections between cells in the same tissue that allow ions and small molecules to pass between cells
• Plasmodesmata


  • Gaps in the cell walls of plant cells that connect adjacent cells and are functionally similar to gap junctions in animal cells
• Cell signaling and signal transduction

  • Cells can both send and receive signals, and respond to external stimuli
  • Stimuli are often small molecules that are recognized by receptors on the cell surface or within the cytoplasm
  • Cells can also respond to physical stimuli like temperature and mechanical forces
  • Receipt of a stimulus results in intracellular signaling events that effect a change in cellular behavior/properties
  • Signaling often occurs through a cascade of events with amplification at each stage
  • Signaling often results in changes in transcription and cell cycle progression
• Development


  Allows a multicellular individual to form from one cell
• Zygote


  Single cell that is a fertilized egg
• Embryo


  Ball of cells formed from the dividing zygote
• Developmental biology


  • Integrates genetics, biochemistry, cell biology, and evolution
• Shared Developmental Processes


  • Cell division
  • Cell-cell signaling and interaction
  • Regulated/differential gene expression (express certain genes and not others)
  • Cell movement or expansion in specific directions
  • Programmed cell death (apoptosis)
• Stem cells


  Undifferentiated cells that keep proliferating
• Asymmetric cell division


  When stem cells divide, they produce a daughter stem cell and another daughter that further divides before differentiating
• Signals


  • Ligands that bind to receptors
  • Binding of signaling ligands to their receptors is very specific
  • Each type of cell expresses a different combination of receptors and is responsive to a different subset of ligands
• Meristems


  Stem cells in plants, present in embryos and adults, produce plant structures throughout a plant's life
• Endocrine signaling


  Signaling molecules are released into the bloodstream and travel to distant target cells
• Somatic stem cells


  Animal stem cells present in most tissues, replace cells that die and repair wounds/tissue damage
• Paracrine & Autocrine signaling

  Signaling molecules act on nearby target cells or the same cell that released them
• Cell-cell interactions in development


  Cells interact through signaling molecules that may diffuse, be present on cell surfaces, or be bound to the extracellular matrix
• Membrane receptor signaling


  Signaling molecules that cannot diffuse across the plasma membrane bind to membrane receptors, triggering a complex series/cascade of events inside the cell to convert the extracellular signal to an intracellular signal and cellular response
• Cell differentiation


  Cells specialize and follow a particular developmental path based on the signals they receive