Meeting 10 & 11

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Meeting 10 & 11
2011-11-08 21:31:20

5.0, 5.2 (5.1! whoops)
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  1. when studying new (newly discovered) proteins, three questions are asked to understand it better:
    • 1) what's it's function?
    • 2) where is it located?
    • 3) what's its structure?

    • can use THREE tools to figure out the answers to these questions:
    • 1) the GENE that encodes the protein
    • 2) a mutant cell line that LACKS the function of the protein
    • 3) some of the actual protein so it can be studied
  2. classical (forward) genetics
    • -start with a mutant defective in some process of interest
    • -isolate the affected gene
    • -isolated gene can be manipulated to produce LOADS of the protein for experimentation
  3. reverse genetics
    same essential steps; reverse the order

    • -begin by isolating a protein of interest
    • -find the corresponding gene (easy [ish] because we have most genome's sequenced)
    • -gene can be altered and reinserted back into an organism to see how the mutation affects it
  4. recessive alleles usually result from a mutation that ________ the affected gene: this leads to a ____ of function

    conversely, dominant alleles usually result from a mutation that causes a ____ of function
    recessive = mostly inactivation of a gene; leads to a LOSS of funciton

    dominant mutations lead oftentimes to a GAIN of function
  5. EMS
    ethylmethane sulfonate; agent commonly used to induce mutations in experimental organisms; does so by modifying G bases; leading to a mutation of G*C pairs to A*T pairs

  6. silent point mutation
    no change in the amino acid sequence of a protein
  7. missence mutation
    point mutation that results in substitution of one amino acid for another
  8. nonsense mutation
    point mutation that results in a premature stop codon, aka in the middle of an amino acid sequence
  9. frameshift mutation
    point mutation that changes the reading frame of the gene
  10. recombinant DNA
    simpy any DNA molecule composed of sequences derived from different sources
  11. most important enzymes for DNA recombinations:
    restriction enzymes and DNA ligaases
  12. restriction enzymes
    endonucleases produced by bacteria that recognize 4-8 bp sequences (restriction sites) and cleave BOTH strands of DNA at these sites
  13. modification enzymes
    • bacteria also produce a modification enzyme for EVERY restriction enzyme produced; these PROTECT a bacteria's OWN genome from cleavage by protecting or modifying at a potential cleavage site
    • -specifically a METYL group is added within 1 or 2 bases of the restriction sequence/site
  14. most restriction enzymes make staggered cuts in the DNA, but these two make the other kind:
    SmaI and AluI generate blunt end cuts

    • ex. SmaI: CCC^GGG
    • GGG^CCC
  15. the frequency at which a restriction enzyme cuts DNA depends on the length of a recognition site; so for ex. if a restriction enzyme recognizes a 8-bp sequence it will cleave DNA once every:
    48 base pairs (~65kb); the fragments that result from such cleavages are called restriction fragments
  16. blunt-end ligation
    DNA ligase from bacteriophage T4 can ligate an two BLUNT DNA ends; but such ligation is inefficient and requires a greater concentration of both DNA and DNA ligase than does sticky end ligation
  17. plasmid vectors must have (at least) 4 regions essential for DNA insertion and subsequent cloning:
    • 1) replication origin corresponding to the HOST
    • 2) marker that permits selection
    • 3) a drug resistant gene (so those vectors that take up the plasmid can be isolated)
    • 4) region that can take up the DNA to be inserted (polylinker)
  18. polylinker
    the 4th region necessary in a plasmid; it's a synthetically generated sequence that has ONE copy of different restriction sites NOT present elsewhere in the vector; when the vector is treated with one of these specific enzymes, it opens up only at one site (with stick ends) and if the DNA that's going to be inserted has also been treated with said enzyme the ends can be ligated together.
  19. genomic DNA library
    an entire genome is fragmented and individual fragments are cloned into a vector
  20. cDNA library
    • 1) mRNA is isolated from cells (easy to distinguish from other types of RNA because polyA tails hybridize to oligo-dT primers!)
    • 2) reverse transcribed into cDNA (mRNA removed using alkali)
    • 3) individual cDNAs are cloned into a vector
  21. reasons why genomic DNA libraries are difficult for higher eukaryotes:
    • 1) introns make the actual genes too long to be inserted into plasmid vectors
    • 2) again, the presence of introns and intergenic regions make it difficult to identify the actual coding sequences (what's important)

    this is solved by using cDNA libraries
  22. hybridization
    the ability of complementary SINGLE-stranded DNA or RNA molecules to associate specifically with each other via base-pairing
  23. most useful oglionucleotide length:
    20 bp; long enough to it's sequence to occur uniquely; occurs every 420 nucleotides; means it usually only occurs once in a genome
  24. shuttle vector
    type of vector capable of replication in two different hosts

    • contains 3 EXTRA elements in addition to all those found in the plasmid vector (so in total it has 6 important regions)
    • 1) ARS: ORI for DNA in yeast
    • 2) CEN: yeast centromere which allows for segregation of the plasmid during yeast cell division
    • 3) URA3: gene that codes for an enzyme that makes uracil; selectable marker
  25. what are the 6 structures necessary in a shuttle vector?
    polylinker, ORI (bacterial), ampr selective gene, CEN, ARS (yeast ORI), and finally URA3 (yeast selective marker for uracil)
  26. functional complementation
    restoring wild-type function by introducing a functional copy of a gene into an organism in which that gene is mutated
  27. incorporation of a ddNTP terminates strand elongation; instead of a 3' hydroxyl group there's just an H atom; cannot form a phosphodiester bond (aka dehydration reaction can't occur) with the next incoming nucleotide
  28. Sanger Method
    blah blah blah
  29. drawbacks of cloning (aka using vectors)
    • 1) it's slow
    • 2) constrained by the size of DNA fragment the plasmid is capable of holding
    • 3) have to have convenient restriction enzyme sites
  30. process of PCR:
    should be able to narrate that video

    • 96---heat denaturing
    • 50-60 --- primer annealing
    • addition of Taq pol (can synthesize DNA even at high temperatures, aka at temperature where the two original strands or subsequently created strands won't reassociate)
    • 72 --- complementary DNA strand synthesis


    it's only after the 3rd cycle that the DNA sequence length of choice is actually present in the mixture