Biochem 501: Part IV.11: Techniques to investigate Genetic information transfer

  1. How to determine between Type I/II recombinase?
    • Mutate Tyr/Ser in gene, observe effects on protein
    • If mutating Tyr stops it= T1, if Ser= T2
  2. Process to apply mutation to hypothetical ENE gene
    • 1. isolate DNA sequence coding for ENE
    •     -PCR to amplify
    • 2. introduce mutations that disrupt tyr or ser residue
    •      -Use synth oligonucleotides w/ mutant sequence to create mutations
    • 3. Join mutant ENE gene with DNA vector
    •     -restriction enzymes and DNA ligase
    • 4. transform bacteria mutant ENE plasmid
    • 5. induce bacteria to make protein, isolate for molecular assays
  3. Generate recombinant DNA bacteria
    • Take gene of intrest
    • Place into a plasmidm with selection gene (antibiotic resistence)
    • Put plasmid into bacteria
    • Now you have a recombinant bacteria with the gene of interest.
  4. Plasmid:
    • DNA polynucleotide capable of independent regulation, contain:
    • mechanism for integration
    • origin of replication (ori) recognized by host machinery
    • (when in research) selection gene
  5. Polymerase Chain Reaction (PCR)
    • PCR amplifies a target DNA sequence via multiple rounds of DNA synthesis
    • Only target DNA is amplified
    •       -Only add short polynucleotide oligonucleotides  to prime a specific gene's DNA polymerization
    • Use DNA polymerase from thermophylic microbe, allows DNA to be made at the high temps needed for DNA denaturement
  6. Three steps of PCR
    • DNA denatured
    • Short polynucleotides prime polymerase
    • Polymerase synthesizes copies of the DNA
    • Repeat
  7. Uses for synthetic DNA
    • Produce the oligonucleotide primers for PCR
    • Introduce specific/random point mutations into recombinant DNA molecules
    • Possible to synthesize entire genes
    • (first synthesized yeast tRNA)
  8. Process of synthesizing polynucleotide chains
    • Attach 1st nucleotide to silica support, prep for next nucleotide (functional groups protected with DMT to prevent accidental reactions, removed after attachment)
    • Next nucleotide added to 5' OH of 1st nucleotide (chemical synthesis is 5'->3')
    • Repeat
    • When done, remove blocking groups from bases, remove methyl groups from phosphates, cleave chain from silica support
  9. Molecular biology commonly uses what?
    Type II restriction enzyme
  10. Type II restriction enzymes are:
    sequence specific (generate products with 5' phosphate and 3' OH)
  11. Uses for Type II restriction enzymes
    • DNA cloning
    • Diagnose presence of mutation if it adds a new/removes restriction site
    • Can be used to identify source of tissue sample
  12. DNA Ligases
    Catalyze Phosphodiester bond formation, DNA ligase acts during replication to seal nicks in lagging strand synthesis
  13. Ending PCR's
    Commonly use ddNTP, these lack an OH at the 3' position, so the reaction ends
  14. Sequencing DNA in a PCR
    • Fluorescent tags added to the ddNTP
    • See how long each strand is with a gel run (high resolution polyacrylamide gel electrophoresis or capillary electrophoresis
    • Shortest is first, look at color, covert to flavor or ddNTP, and go from there.
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Biochem 501: Part IV.11: Techniques to investigate Genetic information transfer
Biochem 501 FINAL