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14 Mystery: The Crooked Cell Upon learning that her Uncle Eli is afflicted with Sickle Cell Anemia, Ava begins to furiously research the disease and her family history to deternmine if she might be at risk for developing the disease or passing it to her children. Ava studies her school textbook to learn more about Uncle Elis condition. She discovers that sickle cell disease is caused by a simple change in the DNA of one gene. The change involves a substitution of the nucelotide Adenine (A) to a Thymine (T) in a gene called Betaglobin. In her Biology class, Ava learned that changing a single nucleotide can change an amino acid in a polypeptide change (Figure 2), but she is confused as to how this single base difference can cause all of her uncles symptoms, so she does a bit more research about the Beta-globin protein. Ava finds that Beta-globin is one of two polypeptides that make up a very important protein called hemoglobin. Hemoglobin is the protein in red blood cells that is responsible for transporting and delivering oxygen to working tissues so that they can carry out the essential process of cellular respiration. Without sufficient oxygen, cells run out of energy to perform their daily work and quickly die. Hemoglobin also helps to transport carbon dioxide, a waste product of cellular respiration, from cells to the lungs. Hemoglobin relies on the efficient diffusion of both oxygen and carbon dioxide to maintain normal body function. Ava discovers that the single nucleotide mutation in her Uncle Eli prevents his hemoglobin from properly carrying oxygen. In addition, the hemoglobin proteins in his red blood cells link together, pulling his cells into the trademark sickle shape found in this disease. The sickle-shaped cells do not flow easily through the tiny capillaries in the body. They can cause blood clots that can be fatal if left untreated. After learning about sickle cell disease and its symptoms, Ava is interested in learning about how Uncle Eli inherited the disease, whether or not she is in danger of developing the disease, and her chances of passing the disease onto her children (after she is married and has a great job). She reads about a biotechnology company that is offering a FREE family sickle cell screening. She immediately gets permission from her parents and gathers as many family members as possible to be screened with her. At the lab are: Ava, her mother, Uncle Eli, Avas grandmother and grandfather (the parents of Uncle Eli and her mom), and her other grandmother (Avas fathers mother). Avas dad is away on a business trip at the time of the testing and cannot participate. Ava doesnt really understand the method used by the biotechnology company, but they provide her family with a handout to explain how they will screen each member for the sickle cell allele.

Screening for Sickle Cell Anemia: An Informational Brochure Thank you for taking the time to participate in our free sickle cell screening. In todays test, we will follow the basic steps below to identify whether you are afflicted by sickle cell disease, a carrier for the disease, or completely free of the disorder. Step 1: Obtaining the DNA we will ask you to take a swab of the inside of your mouth. This is a painless procedure used to collect DNA from each test subject Step 2: Isolate the DNA we will isolate the DNA from your cotton swab and store the DNA in a properly labeled and sterile test tube Step 3: DNA Digestion Using the restriction enzyme Mst II, we will cut the DNA into fragments. The recognition sequence for Mst II is CCT-GAG-GAG. In a normal Beta-globin gene, this sequence is recognized and cut. In a sickle cell gene, the single nucleotide mutation occurs within the recognition sequence and changes it to CCT-GTG-GAG. This simple change alters the recognition sequence and Mst II will not cleave the DNA. See the image below:

Step 4: Visualize Restriction Fragments In order to visualize the DNA and identify the presence or absence of the disordered allele, each DNA sample will be run through a gel electrophoresis apparatus. Gel electrophoresis uses DNAs negative charge to separate fragments of various lengths from each other.

Only the circled fragments between each restriction site, seen in blue will be viewed on the gel. The remaining DNA will not be visualized. Therefore: the Sickle cell DNA will appear as one band, while the homozygous normal DNA will appear as two.

Within the apparauts, large DNA fragments travel a short distance from the origin whereas smaller fragments travel further from the origin. Since DNA is colorless, a stain is applied to the completed gel in order to visualize the different fragment bands. Lab Objective: Your job, as a technician at the biotechnology firm, is to complete the gel eletrohoresis procedure and to determine which of the family members is afflicted with sickle cell anemia, which members are carriers for the disease, and which members are completely disease free. Ava and her family members will have their DNA run on the same gel since they are related. Procedure: 1. Using the micropipetter, and CLEAN TIPS FOR EACH SAMPLE, load the DNA samples A-F into the wells in consecutive order. Lane Tube Person 1 A Avas Grandmother on her Fathers side 2 3 4 5 6 B C D E F Avas Mom Ava Uncle Elis Mother Uncle Eli Uncle Elis Father

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After all of the DNA samples are loaded, connect the apparatus to the power source and set the power source to high.

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Check that the current is flowing properly you should see bubbles forming on the metal wires on either end of the chamber.

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Run the electrophoresis for approximately 20-30 minutes. Be sure to watch the dye. If it gets close to the end of the gel, shut off the apparatus to prevent the DNA from running off the gel. Put on gloves. Remove the tray containing the gel and slide the gel onto a staining tray. Place an InstaStain patch onto your gel (stain side down). Your instructor will complete the staining process. Your gel will be ready to analyze during your next class period.

Pre-Lab Questions: (answer in complete sentences on your student copy of this lab) 1. Using your own words, restate the objective of this lab in the objective section of your student sheet.

Answer Questions 2 7 in complete sentences in the background information section of your student sheet.
2. What is the point mutation that causes sickle cell anemia? Where is it located? 3. How many polypeptides are contained in hemoglobin? How is the polypeptide structurally different in the hemoglobin of individuals who are affected by sickle cell anemia? 4. Describe how the blood disease sickle cell anemia affects its victims. 5. How will the biotechnology company collect and isolate the DNA from Ava and her family members in order to screen for the sickle cell allele? 6. What restriction enzyme will be used to digest the DNA that is collected? How will this restriction enzyme be used to detect the sickle cell allele (be specific). 7. List in 2-3 steps, how the biotechnology company will view the restriction fragments using gel electrophoresis. 8. Summarize the procedure of this lab in 3-5 sentences in the procedure section of your student sheet. Complete a hypothesis by answering these questions in the hypothesis section of your lab handout. 9. What do you think Avas genotype is for sickle cell anemia? Explain! (answer in complete sentences) 10. Draw lines to indicate the DNA patterns on a gel electrophoresis for a person homozygous normal (non-sickle cell anemic), heterozygous, and homozygous recessive (sickle cell anemic) See the blank, labeled gel in the hypothesis section of your student sheet. Analysis: 1. Make an ACCURATE drawing of your results. Be sure to label each sample (A-F, as well as the persons name) as well as the positive and negative sides of the gel. 2. Complete the table based on your results: Lane 1 Tube A Person Avas Grandmother on her Fathers side Avas Mom Ava Uncle Elis Mother Uncle Eli Uncle Elis Father Phenotype Genotype

2 3 4 5 6

B C D E F

Conclusion: (Answere these questions in complete sentences 1. 2. Explain why the carriers genetic profile appeared the way it did. Using the lab results for Ava and Avas grandmother on her fathers side, determine Avas fathers genotype and phenotype for the sickle cell trait. Explain how you came to this conclusion.

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Identify and describe two other applications of gel electrophoresis.