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.

GENE EXPRESSION CENTER

Scanner - DNA chip goes in here

Image on screen as chip is scanned

Smoker vs. Former Smoker

Smoker vs. Non-Smoker

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4 5 6

7 8 9

10 11

Probe DNA(each grid)

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.

A Real DNA Chip Each spot is a different gene

Example:Green Smoker Red Non-smokerYellow Both tissues express the gene

Bioinformatics - It’s a BLAST

Sequences at each spot on the chip are provided on kit CD

BLAST: “Basic Local Alignment Search Tool”

GENE 1 - CTCATCAGTAATGGTCAGAGCAT GENE 2 – GAACAGCTGTTCAGTCTGCAAGT GENE 3 – CTGGGGTTAGTGCTGGCGATCCTGENE 4 – CCAGGAGCCCCAGTTACCGGGAGGENE 5 – GGAGAAGGTAGATTTCAATACGTGENE 6 – CATGATAAGGCTCTTACCCCCTTGENE 7 – GTCATGGCGACTGTCCAGCTTTGGENE 8 – TTGTTTTTGAGACAGAACCTTGCGENE 9 – ATCCTCCAGGTGCACAAGCTCCAGENE 10 – ACCATGGATGATGATATCGCCGCGENE 11 – TGAACGCATTCATCGTGTGGTCT

Probe Gene Sequences - Cancer / Smoking Chip

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)

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 illustrateseveral important biological principles.

http://www.madison.k12.wi.us/west/science/biotech/at-genes.htm

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

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:

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

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.

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