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Large Scale Gene Expression with DNA Microarrays

Large Scale Gene Expression with DNA Microarrays. Vermont Genetics Network Microarray Outreach Program. Vermont Genetics Network (VGN) Founded at the University of Vermont in 2001 through an NIH BRIN grant. Purpose: Encourage biomedical research in Vermont

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Large Scale Gene Expression with DNA Microarrays

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  1. Large Scale Gene Expression with DNA Microarrays Vermont Genetics Network Microarray Outreach Program

  2. Vermont Genetics Network (VGN) • Founded at the University of Vermont in 2001 • through an NIH BRIN grant. • Purpose: • Encourage biomedical research in Vermont • Create a “network” of researchers and students • Give outreach lectures to 4-year institutions • Provide research grants to faculty and students • Mentoring for students interested in research

  3. VGN Microarray Outreach Program • Introduce microarray technology to VT colleges. • Develop microarray outreach module. • Team of scientists to serve as instructors • Ahmad Chaudhry, Rebecca Guy, Tim Hunter, Brian McElhinney, Pat Reed • Kathy Seiler, Scott Tighe

  4. Microarray Experiment • The effect of a chemical dimethyl sulfoxide (DMSO) • on gene expression in yeast • DMSO is an environmental contaminant from the paper industry and from pesticides • Grow the yeast and treat one group with plain water • (control group) and the other group with 10% DMSO • Isolate RNA from the yeast grown in two different conditions, prepare target from it and use it on microarrays to see changes in gene expression

  5. Expressed Genes = mRNA DNA (genes) messenger RNA Protein (effector molecules)

  6. What are Microarrays? • Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of genes (usually between 500-20,000) • Via a conventional DNA hybridization process, the level of expression/activity of genes is measured • Data are read using laser-activated fluorescence readers • The process is “ultra-high throughput”

  7. Why use Microarrays? • What genes are Present/Absent in a cell? • What genes are Present/Absent in the experiment vs. control? • Which genes have increased/decreased expression in experiment vs. control? • Which genes have biological significance?

  8. Why analyze so many genes? • Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function. • Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways

  9. mRNA cDNA cDNA cRNA Creating Targets Reverse Transcriptase PCR amplification of DNA More in vitro transcription

  10. RNA-DNA Hybridization Targets (RNA) probe sets on chip (DNA) (25 base oligonucleotides of known sequence)

  11. Non-Hybridized Targets are Washed Away Targets (fluorescently tagged) “probe sets” (oligos) Non-bound ones are washed away

  12. The 6 steps of a DNA microarray experiment (1-3) • Manufacturing of the microarray 2. Experimental design and choice of reference: what to compare to what? 3. Target preparation (labeling) and hybridization

  13. The 6 steps of a microarray experiment (4-6) 4. Image acquisition (scanning) and quantification (signal intensity to numbers) 5. Database building, filtering and normalization 6. Statistical analysis and data mining

  14. Experimental Design • Choice of reference: Common (non-biologically relevant) reference, or paired samples? • Number of replicates: How many are needed? (How many are affordable?). Are the replicate results going to be averaged or treated independently? Is this a “fishing expedition” or a hypothesis-based experiment?

  15. Why Use Yeast ?? • easily manipulated in the laboratory • simple eukaryote, unicellular • rapid growth (doubling 1.5 - 2.5 hours) • non-pathogenic • stable haploid and diploid states • complete genome sequenced

  16. E. coli ~ 1 x 3 m Yeast ~ 5 m dia. Human ~ 1.7 m 1 chromosome 4 x 10 6 bp 16 chromosomes 12 x 10 6 bp 23 chromosomes 3.3 x 10 9 bp ~ 30, 000 genes ~ 4,377 genes ~ 5,726 genes

  17. Yeast Life cycle

  18. S. Cerevisiae Genome • DNA ~ 60% A + T • 16 chromosomes (haploid) • chromosomes have centromeres and telomeres • also have mitochondrial genome • plasmid: 6 kb, 60-100 copies/cell • entire genome has been sequenced

  19. Yeast Genetic Nomenclature • genes names are 3-letters + a number • genes names are derived from phenotype of the mutant, i.e. ste 3 mutants have sterile phenotype • gene names are written in italics or underlined • wild type alleles: Uppercase STE 3 • mutant alleles: lowercase ste 3

  20. Growth and Metabolism + O2 / no O2 CARBON + NITROGEN + BIOTIN + MINERALS Prototroph: requires no additional nutrients (can grow in minimal media). Auxotroph: requires additional nutrients from the environment or media. Our strain,NRRL Y-12632(orATCC 18824) is a wild type, prototrophic, Matαstrain isolated from brewing yeast.

  21. Changes in gene expression? Which genes are up regulated? Which genes are down regulated? What do the results say about yeast biology? What parallels (if any) can we make to human biology? untreated versus DMSO treated Yeast Microarray Experiment

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