In this lab you will be investigating BRCA to determine whether or not it functions to serve a positive or a negative effect on the body.

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1 BRCA 1 and BRCA 2 are two genes that have been linked to a person s risk for developing cancer and specifically Breast Cancer. When either or both of these two genes are found to be mutated in a person, it can be passed to their genetic relatives. BRCA genes are just two of a large amount of genes that are involved with regulating the cell cycle and the rate at which a cell divides through various checkpoints in a cell s life. In this lab you will be investigating BRCA to determine whether or not it functions to serve a positive or a negative effect on the body. You will be looking for this gene by conducting DNA gel electrophoresis on healthy tissue compared to tissue from a tumor, looking for the presence of the BRCA gene in each. Generally speaking, there are genes that cause cancer known as Oncogenes and there are also genes that control cell growth and protect us from cancer called, Tumor Suppressor Genes. You have deliberately NOT been told anything further, your task is to figure out what kind of gene the BRCA genes are. Think about it 1. Prediction, do you think BRCA 1 and 2 are tumor suppressor genes that protect from cancer or oncogenes that cause cancer? Is BRCA a good gene or a bad gene?

2 Lab Protocol: 2. A DNA ladder, isolated BRCA gene, and three DNA samples have been mini-prepped for you as listed in the table below: 3. A 1% DNA gel has been prepared (0.5 grams of Agarose to 50 ml of 1x LB). First remove the blue comb and then carefully remove the two black rubber bumpers from the gel mold. 4. Insert the gel mold into the electrophoresis chamber and fill the chamber with 1x LB buffer until the gel is just barely submerged. 5. Load each sample into the wells as indicated by the table above. 6. Place the cover on the chamber and carefully bring it over to a power supply set to 300V for 12 minutes. 7. After the gel has run, unplug it from the power supply and carefully carry the entire chamber up to the front lab table so it can be viewed on the UV Transiluminator. Note: Dye has been loaded into your gel that is only excited by UV light NOTES: While your gel is running what s happening? 8. What is the technique of DNA Gel Electrophoresis used for? 9. Genes sort by within a DNA gel. 10. What direction do the DNA fragments travel and why?

3 Post-Lab Analysis: 11. Take a picture of your actual gel and insert it below: Insert image here as a drawing 12. Using either your actual lab group s gel or the stock image to the right, where is the BRCA gene? Is it in the Normal tissue in Lane 3 or is the BRCA gene found in the Tumor Cells in Lane 4? 13. So what can you conclude from your results? Is BRCA a good Tumor Suppressor gene or is it a bad Oncogene that causes cancer? Explain your reasoning. 14. Measure the distance each fragment of the DNA ladder migrated down the gel from the starting line and record in the table provided.

4 15. Plot the points and draw a best fit straight line of your DNA standards on the log graph paper provided. 16. Measure the distance of migration of the known BRCA gene in lane 2. Use your graph and determine from the DNA standard, how long is the nucleotide sequence is of the BRCA gene? Scientists use special enzymes called restriction enzymes that cut at specific DNA sequences. This allows them to find specific regions in the DNA code. In this lab, an enzyme was used to seek out and cut the mutated sequence of the BRCA gene into two pieces. 17. Measure the distance of migration of the 2 smaller fragments that resulted in the cutting the mutated BRCA genes in lane 4. Use your graph and determine from the DNA standard, how long each nucleotide sequence is for the two fragments. 18. Do these fragments sizes add up to the normal BRCA gene length? Should they? If they didn t what might be off? 19. Look at lane three now, based on what bands you see, what can you conclude about how many working copies of the BRCA gene and mutated copies of BRCA gene are present in this person's normal cells. Experimental Design Questions (ACT CRS - Scientific Investigation): 20. Why did we run a DNA ladder in lane 1? What if we did not run a DNA ladder in lane 1, what would we NOT be able to figure out? 21. Why did we run only the BRCA gene in lane 2? What if we did not run the BRCA gene in lane 2? Content Questions Cell Cycle Tumor suppressor genes tell the cell to have Checkpoints at various times during its life. 22. Applying your knowledge of the cell cycle, what does each checkpoint check for?

5 G1 Checkpoint: G2 Checkpoint: Mitosis Checkpoint: 23. Explain the results of lane 5 (the surrounding cells) in terms of the number of good copies of BCRA. Are these cells cancerous? Why or why not?

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