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

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

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

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

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

TopicNext 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

TopicNext 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 orthologsUse sequence to design primers to clone cDNA

Sequencing Genomes1) Map the genome2) Prepare an AC library3) Order the library

4) Subdivide each AC into lambda contigs

5) Subdivide each lambda into plasmids

6) sequence the plasmids

Using the genomeStudying expression of all genes simultaneouslyMicroarrays (reverse Northerns)•Attach probes that detect genes to solid support

Using the genomeStudying expression of all genes simultaneouslyMicroarrays (reverse Northerns)•Attach probes that detect genes to solid support•cDNA or oligonucleotides

Using the genomeStudying expression of all genes simultaneouslyMicroarrays (reverse Northerns)•Attach probes that detect genes to solid support•cDNA or oligonucleotides•Tiling path = probes for entire genome

Microarrays (reverse Northerns)•Attach probes that detect genes to solid support•cDNA or oligonucleotides•Tiling path = probes for entire genome

•Hybridize with labeled targets

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

MicroarraysMeasure amount of probe bound to each cloneUse fluorescent dye : can quantitate light emitted

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

Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”• Fix probes to slide at known locations, hyb with

labeled targets, then analyze data

Using the genomeStudying expression of all genes simultaneously1.Microarrays: “reverse Northerns”2.High-throughput sequencing