1. 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

2. 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

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

4. 1 microliter spots of DNA in solution
Probe DNA
1 microliter spots of DNA in solution
Each grid: 11 genes in duplicate

5. 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

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

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

8. 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

9. 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