Meeting 12

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Meeting 12
2011-11-09 13:46:00

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  1. southern blot
    • 1) restriction fragments are run through gel
    • 2) then denatured by alkali solution and transfered onto nitrocellulose filter (or nylon membrane) via blotting
    • -this process creates a replica of what was on the gel onto to the filter because probes don't readily diffuse into gel
    • 3) filter/membrane is incubated under hybridization conditions with a specific radiolabeled DNA probe
  2. northern blot
    • 1) mRNA fragments are run through gel (NOT cleaved by restriction enzymes, those only work for DNA)
    • 2) mRNA patterning is then denatured by alkali solution and transfered onto nitrocellulose filter (or nylon membrane) via blotting
    • -rest is the same
    • ·add formamide to gel running buffer: prevents mRNA from degrading; also prevents mRNA from forming secondary structures with itself: disrupts hydrogen bonds while it's running allows mRNA to run in a particular line
    • -normally lukemia cells (when uninduced) fail to differentiate into other types of cells
    • -but upon undergoing treatment to differentiate, one can use Northern Blotting to see how much and if any mRNA is being created by the cells
    • -the presence of mRNA indicates differentiation and therefore indeterminate and possibly harmful proliferation will decrease
  3. reverse transcriptase PCR
    oligo-dT primer hybridizes to mRNA polyA tail, used as the primer for the first starnd of cDNA synthesis by reverse transcriptase
    • -use expression vectors to make a lot of protein (useful if it's naturally found in low abundance)
    • -way this is done is by including a promoter native to the organism doing the recplication (in this case, E.coli) as well as the cDNA (aka gene) sequence of interest next to the promoter in the vector
    • -in this system, the lacZ gene is replaced by a G-CSF gene (that makes the desired protein) and the vector is placed in a medium containing a lactose analog IPTG
    • -promoter/vector keeps producing the gene it tHINKS is lacZ but is really G-CSF and therefore lots of protein is made
  4. to expedite the purification process of a protein made in an e.coli expression system:
    • the cDNA to be inserted is often modified at its end with a short nucleotide sequence so that the expressed protein has 6 histidine (basic!/+) residue at the C-terminus that easily binds to an affinity matrix that contains chelated nickel
    • -bound proteins can be released by washing the matrix with a low-pH buffer (aka decreasing the pH of the surrounding medium so the histidine residues are less attracted to the matrix than they are to the medium)
  5. transfection
    how genes are cloned into specialized eukaryotic expression vectors and then introduced into cultured animal cells; recombinant vector can either be integrated into the host's DNA or remain separate
  6. transient transfection
    recombinant vector is NOT integrated into the host's DNA; remains a separately replicating vector; plasmids NOT segregated into daughter cells (inserted into host's cell via electroporation of lipid treatment)

    vectors (similar to yeast shuttle vectors) must contain: ORI from virus, promoter recognized by (mammalian) host's RNA pol II, and a cDNA encoding protein to be expressed next to the promoter
  7. stable transfection
    recombinant vector IS integrated into the host's DNA, therefore passed onto daughter cells when replication occurs
  8. promoter-fusion
    when the promoter for the gene that encodes the protein of interest is linked directly to the coding sequence of a reporter protein (aka GFP); florescence is seen where translation takes place; i.e. where the proteins are located immediately after they are produced (cytoplasm)
  9. protein-fusion
    when the promoter for the gene that encodes the protein of interest is linked toa hybrid consisting of the coding sequence of the protein of interest PLUS the coding sequence of a reporter protein (aka GFP); can be seen more distally from the act of translation into these certain proteins; AKA florescence is seen where hte protein of interest is NORMALLY located in the organism
  10. there are problems with gene therapy
    sometimes inserting a functional gene where there is a mutation can result in proper production of the protein needed; but sometimes this insertion of nucleotides mid-gene can activate an oncogene which leads to cancer (unending proliferation of a certain cell that should have died or just proliferation in general)