1. DNA Chip Scanning DNA Chips are scanned with a laser to excite the fluorescein dye that is attached to the target cDNA. Only those probe spots where target cDNA hybridized will fluoresce.

4. GENE EXPRESSION CENTER

5. Scanner - DNA chip goes in here

6. Image on screen as chip is scanned

7. Smoker vs. Former Smoker Smoker vs. Non-Smoker 1 2 3 4 5 6 7 8 9 10 11 Probe DNA (each grid)

8. DNA CHIP before hybridization with target DNA (after spotting Probe DNA) Sometimes the SDS used in the wash solutions will fluoresce. This is all background signal.

9. A Real DNA Chip Each spot is a different gene Example: Green Smoker Red Non-smoker Yellow Both tissues express the gene

10. Bioinformatics - It’s a BLAST Sequences at each spot on the chip are provided on kit CD BLAST: “Basic Local Alignment Search Tool”

11. GENE 1 - CTCATCAGTAATGGTCAGAGCAT GENE 2 – GAACAGCTGTTCAGTCTGCAAGT GENE 3 – CTGGGGTTAGTGCTGGCGATCCT GENE 4 – CCAGGAGCCCCAGTTACCGGGAG GENE 5 – GGAGAAGGTAGATTTCAATACGT GENE 6 – CATGATAAGGCTCTTACCCCCTT GENE 7 – GTCATGGCGACTGTCCAGCTTTG GENE 8 – TTGTTTTTGAGACAGAACCTTGC GENE 9 – ATCCTCCAGGTGCACAAGCTCCA GENE 10 – ACCATGGATGATGATATCGCCGC GENE 11 – TGAACGCATTCATCGTGTGGTCT Probe Gene Sequences - Cancer / Smoking Chip

25. Gene Ontology Describes three features about a gene: Where its protein product is located in the cell (cellular compartment) What process its protein product is part of (cellular process) The function of that protein product (molecular function)

26. What do these Genes Do? The genes on this chip are not intended to give a complete model of lung cancer, but they can be used to illustrate several important biological principles. http://www.madison.k12.wi.us/west/science/biotech/at-genes.htm

27. Gene Expression - Tumor suppressors; oncogenes Developmental stage-dependent genes Biological Principles: Genetic Polymorphism – Aldehyde dehydrogenase has several different versions (alleles) among human populations Biochemical reaction catalysis – Aldehyde dehydrogenase, Glutathione Peroxidase, Cytochrome P450 metabolize a variety of substances Evolutionary Conservation of Protein Structure – Actin and Slit homolog code for similar proteins in several other species

28. Gene 1 - CYP1A1 cytochrome P450 superfamily of enzymes involved in drug metabolism located in the endoplasmic reticulum. induced in the lung up to 100-fold because of tobacco smoking. Gene 4 - ALDH3A1 - aldehyde dehydrogenase detoxification located in the cytosol. 16 different alleles exists, one predisposes cancer Gene 5 - GPX2 - Glutathione peroxidase detoxification and antioxidant functions. located in the cytoplasm. highly expressed in squamous carcinomas of the lung Smoking increases expression (Up-regulates) these genes:

29. Gene 3 - SLIT2 (Slit Homologue) * Involved in cell adhesion * silenced in many cancers (including lung) * may be a tumor suppressor Gene 7 TP53 - p53 tumor suppressor * guardian of the genome: central role in cell cycle * loss of p53 function allows pre-malignant cells to continue proliferating * found in very low levels in normal cells * located in the mitochondrion and in the nucleolus Quitting smoking is worth it! Off in smokers; On in non- and former smokers

30. Gene 6 - CEACAM6 Human Carcinoembryonic Antigen an oncogene involved in adhesion between cells extracellular matrix Over-expression inhibits cell differentiation and disrupts how cells are organized in lung tissue. UP-Regulated (ON) in smokers and former smokers: It’s better to NEVER START smoking! DOWN - Regulated (OFF) in smokers and former smokers: Gene 9 - CX3CL1 - chemokine ligand tumor suppressor involved in cell adhesion and in inflammation. located on the cell surface, membrane & extracellular compartment.

31. Gene 2 - GPC3 Glypican controls cellular response to damage from external substances may control cell growth and cell death (apoptosis) Decreased expression may represent an early step in cigarette smoke–associated lung carcinogenesis located in the plasma membrane and extracellular matrix. UP-Regulated (ON) in smokers and former smokers: On in smokers and former smokers, OFF in non smokers Gene 8 - CLDN10 – claudin Involved in cell adhesion and inflammation located in the plasma membrane