by Jessica Weber, Ph.D.

• Central Dogma of Molecular Biology

• Enzymes

  • Proteins that function as biological catalysts

    • Catalysts accelerate chemical reactions

  • Convert molecules (substrates) into other molecules (products)

  • Enzymes increase the reaction rate by lowering the activation energy

    • Some enzymes can make their conversion of substrate to product occur millions of times faster

  • Specificity comes from each enzyme’s unique 3D structure

    • An enzyme’s activity decreases markedly outside its optimal temperature and pH
    • Many enzymes are permanently denatured when exposed to excessive heat, losing their structure and catalytic properties

    

    

    Video - DNA Structure

    https://www.youtube.com/watch?v=o_-6JXLYS-k

    Structure of DNA & RNA - nucleotides

    

    Structure of DNA & RNA - bonds

    

    DNA vs RNA

    

    Video: The Central Dogma

    https://www.youtube.com/watch?v=gG7uCskUOrA

    Genetic code

    

    Prokaryotes & Eukaryotes

    

    

    Prokaryote & Eukaryote genome organisation

    

    

    Gene organisation

    

    Human genome - 3.2Gb (haploid)

    

    Alleles

    • An allele is one of two or more altenative forms of a gene that arise by mutation and are found at the same place (locus) on a chromosome.

    

    PCR & Sequencing

    Evolution as a toolkit

    • Modern molecular biology and synthetic biology starts with the tools that nature has provided
    • Modifications are then engineered into these starting blocks that allow you to generate products of interest
    • Examples:
      • Polymerase Chain Reaction (PCR) uses the machinery from DNA replication to enable you to exponentially copy a DNA region of interest
      • DNA sequencing uses the machinery from DNA replication with chemical modifications that enables you to discover the order of bases

    Video - DNA replication

    https://www.youtube.com/watch?v=TNKWgcFPHqw

    

    Example Workflow: PCR & Sanger Sequencing

    • Goal: learn the DNA sequence of a specific gene within an individual or organism of interest, then compare the sequence with those from other individuals/organisms.

    • Methods:

      • Amplify DNA fragments of interest using PCR
        • Forward and reverse primers will be designed upstream of the region that will be amplified
      • Sequence your PCR products using Sanger sequencing
        • Compare your sequence to those of other individuals/organisms
          • Sequence alignment
          • Query a database (BLAST)

    Polymerase Chain Reaction (PCR)

    https://www.youtube.com/watch?v=2KoLnIwoZKU

    PCR amplification of target DNA region

    • Repeated cycles of heating and cooling to allow different temperature-dependent reactions.
      • DNA melting
      • enzyme-driven DNA replication
    • Template DNA + reagents
      • primers (short single-strand DNA fragments that have complementary sequences to the target DNA region)
      • dNTPs
      • buffer
      • heat-stable DNA polymerase

  

  PCR & DNA replication machinery

  

  Going back to the start, in the above case, the subtrate is dNTPs, the enzym is DNA polymerase (taq polymerase because it’s heat stable) and the product is the amplified DNA.

  Video: Capillary Sequencing

  https://www.youtube.com/watch?v=KTstRrDTmWI