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Topic Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert. Topic Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert. Topic

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slide1

Topic

Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert

slide2

Topic

Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert

slide3

Topic

  • Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert
  • Next step: deciding which genes to clone
  • Problem = correlating enzymes with genes
slide4

Topic

  • Next step: deciding which genes to clone
  • Problem = correlating enzymes with genes
slide5

Topic

  • Next step: deciding which genes to clone
  • Problem = correlating enzymes with genes
    • Who matches the pH?
    • Who localizes where?
    • Which isoform if alternatively spliced?
slide6

Topic

  • Next step: deciding which genes to clone
  • Problem = correlating enzymes with genes
    • Who matches the pH?
    • Who localizes where?
    • Which isoform if alternatively spliced?
  • Clone several, using one known to work to find orthologs
slide7

Topic

  • Next step: deciding which genes to clone
  • Problem = correlating enzymes with genes
    • Who matches the pH?
    • Who localizes where?
    • Which isoform if alternatively spliced?
  • Clone several, using one known to work to find orthologs
  • Use sequence to design primers to clone cDNA
slide8

Sequencing Genomes

1) Map the genome

2) Prepare an AC library

3) Order the library

4) Subdivide each AC into lambda contigs

5) Subdivide each lambda into plasmids

6) sequence the plasmids

slide9

Using the genome

  • Studying expression of all genes simultaneously
  • Microarrays (reverse Northerns)
    • Attach probes that detect genes to solid support
slide10

Using the genome

  • Studying expression of all genes simultaneously
  • Microarrays (reverse Northerns)
    • Attach probes that detect genes to solid support
      • cDNA or oligonucleotides
slide11

Using the genome

  • Studying expression of all genes simultaneously
  • Microarrays (reverse Northerns)
    • Attach probes that detect genes to solid support
      • cDNA or oligonucleotides
      • Tiling path = probes for entire genome
slide12

Microarrays (reverse Northerns)

    • Attach probes that detect genes to solid support
      • cDNA or oligonucleotides
      • Tiling path = probes for entire genome
    • Hybridize with labeled targets
slide13

Microarrays

    • Attach cloned genes to solid support
    • Hybridize with labeled targets
    • Measure amount of target bound to each probe
slide14

Microarrays

Measure amount of probe bound to each clone

Use fluorescent dye : can quantitate light emitted

slide15

Microarrays

  • Compare amounts of mRNA in different tissues or treatments by labeling each “target” with a different dye
slide16

Using the genome

  • Studying expression of all genes simultaneously
  • Microarrays: “reverse Northerns”
    • Fix probes to slide at known locations, hyb with labeled targets, then analyze data
slide17

Using the genome

Studying expression of all genes simultaneously

Microarrays: “reverse Northerns”

High-throughput sequencing