What does Smoking do to my Genes? DNA Chips (Microarrays - see Animation) Lab Experiment What does Smoking do to my Genes? Observe lung tissue from smokers and non-smokers

LAB Overview 1. Print DNA Chip 1.5. (Teacher) Bake (20-80 min ~ 80°C) 2. Wash (4 x 2 min in SDS and water) 3. Hybridize with Target DNA (room temp ~ 20 min) 4. Wash (4 x 2 min in SDS and salt solutions) 5. Send to Scanning Facility 6. Analyze - bioinformatics tools

Lab Step 1 - DNA Chip Printing Make a DNA Chip Lab Step 1 - DNA Chip Printing

1 microliter spots of DNA in solution Probe DNA 1 microliter spots of DNA in solution Each grid: 11 genes in duplicate

DNA Chip Terminology Probe DNA - short pieces of single stranded DNA attached to glass Target DNA - cDNA from cells grown under different conditions Floating in solution on top of probe DNA example: cDNA from seedlings grown in light vs. dark

Probe DNA - attachment to Glass Slide Treated slide From Telechem International

Probe (Chip) DNA + Target cDNA (Simulated) Hybridization Probe (Chip) DNA + Target cDNA (Simulated) ~20 minutes room temp

Microarray Technology Considerations Costly One Array ~ $400. Not including Tissue Preparation One array can cost >$1000. Scanning equipment >$50-200,000. Complicated - each step requires controls for validation and replicates for reliability Harvesting Tissue Preparing Chip and Tissue Hybridizing Analyzing Software is complicated and expensive Huge data sets Requires sophisticated statistical analysis

WE NEED HIGHLY SKILLED PEOPLE! Physicists Develop instrumentation Chemists develop chip printing, target labeling and Hybridization Biologists Tissue growth and harvesting; interpretation of results Computer scientists and statisticians software development and validation