PopGen_II_ProtocolandDatasheet_

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Feb 20, 2024

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Name: _______________________ Section: ______________________ Lab 2: Population Genetics II Learning Objectives  Calculate allelic and genotypic frequencies in a population.  Determine the expected genotypic frequencies of a population.  Explain Hardy-Weinberg Equilibrium and why it is used in population genetics.  Describe the effects of the five processes (natural selection, sexual selection, genetic drift, gene flow, and mutation) on genotypic and allelic frequencies in a population.  Mathematically demonstrate how each process alters frequencies in a population. Lab Safety  Closed-toe shoes are required  Goggles are required.  Gloves are required.  Do not touch the electrophoresis chamber while the power supply is turned on.  All tips that come into contact with blood/hemoglobin should be disposed of in the biohazardous tip waste.  Remove gloves before using a computer and before leaving the lab room. Required Activities and Assessments Within this lab module, you will need to: 1. Read Biology 2e Free Textbook Chapter 19 2. Read the lab activity in its entirety. 3. Complete the Canvas lab quiz before 7:59 am Monday before lab. 4. Complete all data tables, and write responses to the questions and prompts in the Lab Protocol and Datasheet. Turn in your data tables and responses for the questions, and the text of your written responses, and turn it all in to. Lab Activity 1: Malaria and Sickle Cell Anemia The World Health Organization (WHO) has established that someone dies from malaria approximately every 40 seconds. Since the highest mortality rate is in Africa, WHO has decided to concentrate the bulk of its control budget to Africa. However, Africa is a big place. Your class has been asked to help establish where the eradication efforts should be concentrated. Each lab bench will evaluate blood samples from one of three regions in Africa. You will compare the percentage of sickle-cell carriers for the regions and, based upon the correlation between the prevalence of the sickle-cell gene and malaria, make a recommendation as to where to spend the WHO funding to help the most people. The three regions are: Al-Khartum, Sudan and environs (population 5,274,000). Johannesburg, South Africa and environs (population 7,860,780). Kinshasa, Democratic Republic of the Congo and environs (population 7,273,900). Revised Fall 2023 1

Name: _______________________ Section: ______________________ Each lab bench constitutes a research group that will examine blood samples using gel electrophoresis. Recall from Biology 111 that gel electrophoresis uses an electrical current to run DNA or protein samples through a gel matrix and separate mixed constituents based on molecular size. 1. Retrieve one gel tray, an electrophoresis chamber, and a well comb. Raise the gel tray end gates and tighten the set screws. If an end gate is missing, then use masking or labeling tape to secure the end of the tray. 2. Use a hand protector and obtain a flask of hot 1.3% agarose from the incubator. Pour enough agarose (-35 ml) into the gel tray until the agarose is just below the center notch on the tray. Place a 12-well comb in the outer tray notches and let it set (-20 minutes). 3. Practice using a micropipettor while the gel sets: Obtain an agar plate from the refrigerator and place it in a culture dish. Pour enough water over it to just fill the wells and cover the plate. This simulates the conditions you will face when loading blood samples into the gel. Obtain a 2-20 µI micropipettor and observe the volume scale (Figure 1). Locate the decimal point and adjust until the volume reads 12.0 µI. Push the tip of the micropipettor firmly into a sterile yellow pipette tip to seat the tip. Open a tube of practice loading dye and depress the plunger on the micropipettor to the first stop. Make sure the yellow tip is seated, and insert the micropipettor about 2-3 mm into the sample. Slowly release the plunger to aspirate the dye into the tip. Withdraw the pipette taking care not to touch the sample tube. Hold the micropipettor steady with the tip just over or barely into the target well. Brace your wrist if necessary. Depress the plunger to the first stop and then to the second stop to release the sample. The dye is denser than water so you should see it fall into the sample well. Avoid touching the gel with the tip. Hold the micropipettor over the tip discard container and depress the ejector button to release the tip. If more practice is needed, change tips and repeat this process. 4. The gel is ready if it is slightly cloudy when checked against a dark background. If the gel is cloudy, gently pull the comb straight up to remove. Take care not to break the Revised Fall 2023 2

Name: _______________________ Section: ______________________ wells when you remove the comb. Hold the tray level so the gel doesn't slide off and loosen the set screws to drop the end gates. 5. Center the gel tray on the platform in the electrophoresis chamber. Position the tray so the wells are on your left (align with the black electrode). This assures the samples will run from the negative to the positive electrode during the electrophoresis run. 6. Place the chamber on a dark background within reach of the power supply. Add running buffer to fill both reservoirs to the center platform. Slowly add additional buffer until it fills the wells and barely covers the gel. 7. Retrieve a tray of blood samples from the freezer that contains a set of blood samples. The tray is labeled with the African location. Samples are color coded by region and numbered 1-8. In addition to patient samples, there are two control tubes in a different color. The positive control is labeled "+" and will show a homozygous HbS/HbS genotype for sickle-cell anemia. The negative control is labeled "-" and will show a homozygous HbAHbA genotype for normal hemoglobin. Blood samples are negative for HIV and Hepatitis B. However, human fluids should be considered potential biohazards and appropriate precautions observed. Wear gloves, and properly discard tips, gloves, and empty tubes. Should you spill a blood sample, wear gloves and clean it up with a 10% bleach solution. 8. Set the micropipettor to 12 µI and seat a sterile yellow tip on the pipettor. One student aspirates 12 µI of the positive control sample and loads it into well #1. A second student seats a yellow pipette tip on the micropipettor, aspirates 12 µI of the negative control sample, and loads it into well #2. 9. Each student at the lab bench loads at least one blood sample from the set. Change tips between samples and load 12 µI into an open well. Record the sample tube number for each lane in Table 1-3. 10. Align the lid with the electrodes and use both hands to push it in place. Do not try this one-handed or you may bend the electrodes! Plug the color-coded electrode cables into the power supply. Set the power supply to minimum volts and turn the dial to the left. DO NOT TURN IT ON! Ask your instructor to check the setup. Once you have instructor approval, turn on the power supply and adjust the voltage until it reads 100 volts. 11. Observe the gel to be sure samples are moving toward the positive electrode. Allow the gel to run for 45 to 90 minutes. While the gel is running, complete Lab Activity 2. Revised Fall 2023 3

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Name: _______________________ Section: ______________________ Gel Analysis Check the gel for band separation after 45-60 minutes. If separated, turn off the power supply. Unplug the electrodes and slowly remove the lid by pulling it straight off. Take care not to bend the electrodes as you remove the lid. Carefully remove the gel tray. Hold it upright, raise the end gates and tighten the set screws. Gently pour off any residual buffer solution. 12. Place the tray on a white surface and observe the bands. Brownish bands are hemoglobin. Homozygous samples have one band. Heterozygous samples have two bands. Normal hemoglobin is smaller than sicklecell hemoglobin and travels farther from the well. 13. Record the distances the positive control in lane #1 and the negative control in lane #2 moved from their respective wells. Enter these data in Table 3. 14. Measure the distance(s) the other bands moved. Samples with two bands represent a heterozygous genotype (HbS/HbA). Record the genotype of each sample. 15. Calculate the percentage of samples for each region that have a sickle-cell allele. Positive samples may have the homozygous HbS/HbS or the heterozygous HbS/ HbA genotypes. Enter the calculation in Table 3. 16. Record the data from your group on the class overhead and transfer the other class data to Table 3. Compare the relative percentages of sickle-cell alleles among the regions. Revised Fall 2023 4

Name: _______________________ Section: ______________________ DELETE THE ABOVE MATERIAL AND ONLY SUBMIT THE FOLLOWING PAGES!! Population Genetics II Datasheet Lab Activity 1: 1. Observe the class data. Which region has the highest percentage of individuals carrying a sickle-cell allele? 2. Consider the relationship between the prevalence of the sickle-cell trait and the probability of contracting malaria. Rank the three African regions from most likely to least likely to be exposed to malaria. Most likely: Intermediate: Least likely: 3. Based on the class results, where would you recommend the World Health Organization spend their malaria abatement funds? Explain. Refer to Figure 1 below and note that Anopheles mosquitos are more common in the darker shaded regions and less common in the lighter shaded areas of Africa. These mosquitos transport the Plasmodium parasite so there is a direct correlation between the presence of the mosquito and malaria infection rates. Revised Fall 2023 5

Name: _______________________ Section: ______________________ 4. How do the relative percentages of sickle-cell/ traits in these locations compare to the relative abundance of Anopheles mosquitos? 5. As the malaria death rate increases, what would you expect to happen to the number of sickle-cell/ deaths? 6. Explain your answer in terms of natural selection and the "heterozygote advantage." 7. The sickle-cell allele most likely originated when a mutation occurred in a normal hemoglobin allele. If the trait arose in Africa, where did it most likely originate? Explain. Revised Fall 2023 6 Figure 2. Risk and incidence of malaria. Refer to the map posted in the lab and enter the approximate locations of Al-Khartum, Johannesburg, and Kinshasa on the figure above. Figure 1. Anopheles mosquito zones. Darker coloration indicates a wetter environment.

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Name: _______________________ Section: ______________________ Lab Activity 2: Basic Microscopy 1. What is the total magnification of your microscope when using a 40X objective? 2. Observe the demonstration slide of the Plasmodium parasite and sketch it below. 3. Use your microscope to observe the normal and sickle cell blood specimen and sketch them below. Revised Fall 2023 7

Name: _______________________ Section: ______________________ Sample Lane Distance (cm) Genotype Sample Lane Distance (cm) Genotype Sample Lane Distance (cm) Genotype + control + control + control - control - control - control % with sickle-cell allele= % with sickle-cell allele= % with sickle-cell allele= TABLE Revised Fall 2023 8

PopGen_II_ProtocolandDatasheet_

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