Activity 3.1.4 - DNA Microarray Mrs. Stewart Medical Interventions Central Magnet School

Essential Question How does a DNA Microarray illustrate the differences in gene expression between two tissue samples?

Introduction Genome = 20-25,000 genes DNA Chip or Gene Chips = DNA Microarray Technology If all cells in the body (with a few exceptions) contain the same DNA and genes, what makes a skin cell become a skin cell instead of a lung cell? Answer: gene expression. Which genes are “turned on” or “turned off”

How does a DNA Microarray Work? DNA Microarray Video – Kahn Academy Side note: this video says wells are lined with mRNA complements – but for our purposes they are lined with cDNA complements DNA microarrays work by measuring the amount of mRNA for every gene that is present in a cell sample so that scientists can determine which genes are turned on and which are turned off.

Assignment Create a flowchart for the steps in a DNA Microarray in your lab journals

DNA microarray flowchart Print single-stranded DNA gene sequences onto a microarray (slide) Isolate mRNA from normal and “experimental” cells Prepare flourescent (labeled) cDNA from each of the mRNA samples

DNA microarray flowchart Hybridize (place labeled cDNA sequences onto printed slides with known genes – complementary sequences will bind) Visualize results Analyze

What can we infer from a microarray?

Grandpa Joe Grandpa Joe, Judy Smith’s father, has been a smoker for the past thirty years. Last year, Grandpa Joe came down with a cold that turned into pneumonia. It took him more than a month to recover. The family is very concerned he is going to develop lung cancer. They heard about a study being conducted at the local hospital that is exploring lung-cancer associated genes in smokers and non-smokers. The family convinces Grandpa Joe to participate in the study in order to learn more about his risk for developing lung cancer.

DNA Microarray Lab/Activity The study is investigating six genes thought to be involved with lung cancer using DNA microarray technology. The researchers hope to compare gene expression of the six genes of interest between smokers and non-smokers in order to gain more knowledge of what causes a normal lung cell to become cancerous. You have been assigned to the study. Your first task is to learn more about the six genes of interest.

Predictions Gene 1: Human carcinoembryonic antigen (CEACAM6) This gene codes for a protein that is located in the extracellular matrix. This protein is involved with adhesion between cells and is thought to be a proto-oncogene and when over-expressed is an oncogene. Gene 2: Surfactant protein B (SFTPB) This gene codes for an extracellular protein. This protein enhances the rate of spreading and increases the stability of pulmonary surfactant, a lipid-rich material that prevents lung collapse by lowering surface tension at the air-liquid interface in the alveoli of the lungs.

Predictions Gene 3: P53 tumor suppressor (TP53) This gene codes for a protein that is located in the mitochondria and in the nucleolus. This protein is involved with cell cycle checkpoints. This gene is a tumor suppressor gene and is thought to be the “Guardian of the Genome.” Gene 4: SRY This gene codes for a protein that is located in the nucleus. The protein that this gene codes for is testis-determining factor (TDF) which initiates male sex determination. This protein has no function in lung cells.

Predictions Gene 5: Cytochrome P450 (CYP1A1) This gene codes for a protein that is located in the endoplasmic reticulum. The protein catalyzes reactions involved in drug metabolism and synthesizes cholesterol, steroids, and other lipids. The expression of this protein is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. Gene 6: Glypican 3(GPC3) This gene codes for a protein that is located in the plasma membrane and extracellular matrix. The gene controls cellular response to damage and may control cellular growth regulation and apoptosis. This gene is considered to be a tumor suppressor gene for lung cancer.

Procedures Place one drop from each gene onto the correspondingly numbered spot on the microarray slide These will harden in one minute These spots represent the single-stranded DNA from each of the genes

Procedures Obtain a cDNA dropper bottle (Hybridization Buffer) and place one drop onto each of the numbered spots on the microarray slide Careful!! Do not touch the tip of the bottle to the DNA spots The cDNA dropper bottle contains a solution of labeled cDNA from Grandpa Joe’s lung cells and a non-smoker’s lung cells mixed together

Procedures Place your DNA microarray slide onto a white piece of paper to observe results. After you complete your student response sheet: Wipe off the six spots on your slide with a paper towel. Wash and dry your slide.

Analysis Draw a diagram of the slide in your laboratory journal. Make sure to clearly indicate which gene is on which spot. Draw your results on the Student Response Sheet. Include a description of the color of each spot. Analyze the results.

Genes being studied Human carcino - embryonic antigen (CEACAM6) This gene codes for a protein that is located in the extracellular matrix. This protein is involved with adhesion between cells and is thought to be a proto-oncogene and when over-expressed is an oncogene. Surfactant protein B (SFTPB) This gene codes for an extracellular protein. This protein enhances the rate of spreading and increases the stability of pulmonary surfactant, a lipid-rich material that prevents lung collapse by lowering surface tension at the air-liquid interface in the alveoli of the lungs. P53 tumor suppressor (TP53) This gene codes for a protein that is located in the mitochondria and in the nucleolus. This protein is involved with cell cycle checkpoints. This gene is a tumor suppressor gene and is thought to be the “Guardian of the Genome.” SRY This gene codes for a protein that is located in the nucleus. The protein that this gene codes for is testis-determining factor (TDF) which initiates male sex determination. This protein has no function in lung cells. Cytochrome P450 (CYP1A1) This gene codes for a protein that is located in the endoplasmic reticulum. The protein catalyzes reactions involved in drug metabolism and synthesizes cholesterol, steroids, and other lipids. The expression of this protein is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. Glypican 3(GPC3) This gene codes for a protein that is located in the plasma membrane and extracellular matrix. The gene controls cellular response to damage and may control cellular growth regulation and apoptosis. This gene is considered to be a tumor suppressor gene for lung cancer. Genes being studied

Reading the Results: Pink result = Gene transcription (expression) is induced Blue result = Gene transcription (expression) is suppressed Purple result = Gene transcription (expression) is not affected Clear/white result = Gene not expressed **These colors differ from a regular DNA microarray in which Red, Yellow and Green are used.

Moral Monday If a pap smear shows “abnormal” cell growth – should the doctor clearly say the words “precancerous” or “cancerous”, even if a simple procedure can eliminate them?

Quantifying the Results When the ratio is greater than one, the gene is induced by tumor formation. This means that the gene transcription was more active in cancer cells (Smoky Joe) than in normal cells. When the ratio is less than one, the gene is suppressed by tumor formation. This means that the gene transcription was less active in cancer cells (Smoky Joe) than in normal cells. When the ratio is equal to one, the gene is not affected by tumor formation. This means that the gene transcription was the same in cancer cells as it was in normal cells. When the ratio is zero, the gene is not expressed in either cell.

Conclusion Questions: How does a DNA Microarray illustrate the differences in gene expression between two tissue samples? Explain the results to Grandpa Joe and his family so that he understands what they mean as far as his risk for developing Cancer. (you must use examples from the results obtained)