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

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

  3. 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

  4. Topic • Next step: deciding which genes to clone • Problem = correlating enzymes with genes

  5. 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?

  6. 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

  7. 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

  8. 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

  9. Using the genome • Studying expression of all genes simultaneously • Microarrays (reverse Northerns) • Attach probes that detect genes to solid support

  10. Using the genome • Studying expression of all genes simultaneously • Microarrays (reverse Northerns) • Attach probes that detect genes to solid support • cDNA or oligonucleotides

  11. 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

  12. Microarrays (reverse Northerns) • Attach probes that detect genes to solid support • cDNA or oligonucleotides • Tiling path = probes for entire genome • Hybridize with labeled targets

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

  14. Microarrays Measure amount of probe bound to each clone Use fluorescent dye : can quantitate light emitted

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

  16. 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

  17. Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” High-throughput sequencing

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