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