Microbiology Module 9

  1. Restriction Enzymes (Slide 2)
    Part of microbial immune system.Patrol microbial DNA. Make DS break at specific sequences (GAATTC) in invading DNA and destroy it.

    Microbes modify their own DNA so that it is immune to REs within their own cells

    Each RE recognizes a specific 4-8 base pair palindromic sequence
  2. Restriction Enzymes Cont. (Slide 3)
    REs are used to cut and paste (ligate) DNAs from different sources --> produces recombinant DNA
  3. Making recombinant DNAs using REs (Slide 4)
    DNA is cloned when it is isolated from its original source and inserted into a vector that can be propagated in a host

    Vectors are self-replicating DNAs (plasmids or phage) that also contain elements that allow manipulation and/or expression of cloned genes
  4. Recombinant DNA cloning vectors (Slide 5)
    DNAs are inserted into vectors, then vectors/inserts are propagated in host microbial cells (yeasts, bacteria)

    Plasmids used to clone small pieces of DNA. Bacteriophage used to clone bigger pieces of DNA ( 9-25 Kbases)

    Plasmid-derived vectors are introduced by transformation into artificially competent (CaCl2 or electroporation) microbial hosts

    Phage or viral DNAs are packaged (in vitro) and then used to infect host cells
  5. Plasmid Vectors (Slide 6)
    Origin of Replication (OriP) used to maintain high copy number (often 100s in a cell) or low, as required

    MCS (Multiple Cloning Site) has multiple RE sites that are present only at this site. Used as cloning sites for genes

    As only 1% of cells are competent for plasmid transformation, selectable markers and selective media are used to facilitate screening

    Other elements: Gene expression, shuttle vector features
  6. Making cDNA (Complementary DNA) (Slide 8)
    mRNA cannot be directly cloned, so functional mRNA (introns removed) from eukaryotic cells is first converted to cDNA using reverse transcriptase and dT primers

    Resulting DS-DNA is then cloned by adding artificial RE sites to the ends and inserting into a vector

    cDNAs from any source can be expressed as proteins when cloned into expression vectors containing proper transcription and translation genetic elemetns
  7. PCR (Polymerase Chain Reaction) (Slide 9)
    Most widely used diagnostic technique in biological science

    Amplifying via DNA replication the sequence of one particular fragment of DNA

    Amplification of DNA is 10^6--10^9X for one round

    Some substances inhibit thermophilic DNAPs and give false negatives

    False positives (most often) because of extreme sensitivity of technique (Single cell's gene can be detected) and cross-contamination via aerosol

    PCR'd genes can be cloned, sequenced, expressed
  8. PCR Cont. (Slide 10)
    Template (can be any source of DNA) is heated to denature

    Primers are short ssDNAs that bind at ends of denatured template (RE sites can be added) as temp is decreased

    DNA is replicated at high temps using DNAPs from thermophiles and dNTPs

    Repeat 20-30 cycles takes 1-3 hours

    Currently these reactions are done with thermocyclers but field versions are being developed
  9. Sequencing DNA
    Each sequencing machine can sequence 10^6 b/day. Provide lots of info. Can allow for microbial genome to be sequenced rather quickly

    With shotgun procedures, whole genomes are randomly sheared and sequenced, then the overlapping sequences are assembled by a computer
Card Set
Microbiology Module 9
Module 9