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Deirdre Simmons

November 7, 2017

Prof. Biardi

Wednesday Evening Lab

Sickle Cell Anemia Lab Report

Introduction

Sickle cell anemia is a disease that causes red blood cells to form into a rigid, crescent shape, causing them to be unable to move as effortlessly throughout the circulatory system, this disease is also known as drepanocytosis (1). This is a hereditary disease that affects many people of the African, Mediterranean, and Indian descent. It affects hemoglobin negatively, which is a transporter of oxygen in red blood cells (2). A mutation forms in the amino acid sequence and function in the hemoglobin, and this eventually results in sickle cell anemia (3). Africa’s population has a very high infection rate of malaria. Carriers of sickle cell anemia, however, have a higher chance of being immune to malaria. An allele in the sickle cell anemia protects the carrier from malaria, which makes it more of a blessing to be a carrier of sickle cell anemia in places like Africa where malaria is very common (4,5). This disease is autosomal recessive, meaning the offspring only affected both recessive alleles (6). Sickle cell anemia affects chromosome 11, on the nucleotide with the sixth codon of the gene. There would be a switch in amino acids, from an A to a T (5,7). Samples containing a PCR-amplified ß-globin gene that had been incubated with MST II were tested for sickle cell anemia. Individual alleles were digested with MST II, which is a restriction enzyme that could recognize special DNA sequences called palindromes. The allele HbA was cut by MST II, and the allele HbS was not cut by MST II. HbA was the normal beta-globin allele, and HbS was the sickle beta-globin allele. Wells must have been closest to the negative electrode since the DNA samples, which included a glycerol gel buffer and Cresol Red, would migrate through the agarose gel towards the positive electrode. Gel electrophoresis is used to help find the size, and therefore the genotype, of the DNA. The DNA fragments mixed with the buffer and tracking dye are injected into the wells made in the gel. After about forty-five minutes, the fragments would have migrated with help from the electrical current, and the results could be interpreted by a picture taken under the blue light (5). Since sickle cell anemia is a recessive trait, we figured our hypothesis would be: The mother will be homozygous recessive, affected by sickle cell anemia. The father will be heterozygous. The genotype of their offspring will be 1:1. One child will be homozygous recessive, affected by sickle cell anemia, and the other will be heterozygous, a carrier of sickle cell anemia. Figures 1 and 2 represent the hypothesis in a Punnett Square and table form.

Figure 1

Rr Rr

rr rr

Punnett Square of Hypothesis

Figure 1 shows the hypothesis displayed in a Punnett Square

Figure 2

Mother Father Child 1 Child 2

rr, homozygous recessive Rr, heterozygous rr, homozygous recessive rr, homozygous recessive

Table Representing Hypothesis

Figure 2 represents the hypothesized genotypes and phenotypes of each family member within a table.

Methods

Sample Preparation

DNA samples of a family of four, from an African descent, were taken to be tested to match with DNA samples of humans who were affected, unaffected, or a carrier of sickle cell anemia. Since HbA was cut when it was incubated with the MstII enzyme, and the HbS was not because MstII did not recognize it (5), the size difference of each product was easily recognizable. To diagnose these alleles with mutations, PCR amplification of the DNA sequence is used (5). The DNA samples of the family of unknown sickle cell traits were mixed with a gel buffer that contained glycerol, and a red tracking dye called Cresol Red.

Gel electrophoresis

1.0% of agarose gel with added SybrSafe Stain was poured into the center of the gel tray. The SybrSafe Stain allowed the DNA to be seen when photographed under the blue light. The gel had to sit for at least twenty minutes, or until the gel had solidified. The gel loading buffer with glycerol caused the DNA samples to sink into the well plates more easily, and the Cresol Red helped the DNA be more visible, making it easier to track (5).  After the gel solidified, for about thirty minutes, we repositioned the casting tray, so the wells were at the negative electrode. We filled the tank with 1X TBE buffer so it just covered the gel. We then removed the well comb. After, we loaded 15 uL of each DNA sample into the well plates. Then, we inserted the plugs of the black electrode lead into the black input of the power source, and the red into the red. After, we set the power source to ~100V and ran the electrophoresis. We let the tracking dye migrate for about forty-five minutes to make sure it was about 3 cm away from the wells. After the electrophoresis was completed, we connected the leads, and removed the cover.

Gel Imaging

We then exposed the gel to the blue-light and took a photo of it using an iPod.

Results

DNA samples of an African American family were analyzed along with controlled DNA samples. Gel electrophoresis was used to determine the genotype of each individual. Figure 1 shows the Punnett Square made based off of the hypothesis. Figure 3 (below) shows the units of gel electrophoresis analyzed. Lane 3 was DNA sample A and part of the control group. It had one thick DNA fragment, that did not migrate very far. It is closer to the negative electrode side. This band represented an individual who was homozygous for HbS, meaning that it was homozygous recessive, affected by sickle cell anemia. Lane 4 was DNA sample B and also part of the control group. It had two thin DNA fragments, one migrated to about the same place as DNA fragment A, and the other was a few millimeters farther, closer to the positive electrode side. These bands represented an individual who was heterozygous, a carrier of sickle cell anemia. Lane 5 was DNA sample C, part of the control group as well. It had two DNA fragments, one thick and closer to the positive electrode side, and one very faint and thin, closer to the negative electrode side. These bands represented an individual who was homozygous for HbA, which meant it was unaffected by sickle cell anemia. Lane 6 was DNA sample D, which was a sample taken from the Mother’s DNA. It had two thin DNA fragments. One migrated a shorter distance, so it was closer to the negative electrode side, and the other migrated a longer distance, which made it closer to the positive electrode side. Compared to the three controlled samples, this DNA sample was most similar to sample B which would mean the mother was heterozygous, a carrier of sickle cell anemia. Lane 7 was sample E, which was the Father’s DNA sample. It also had two thin DNA fragments, making it very similar to both sample D and sample B. One fragment migrated farther than the other, this was closer to the positive electrode, the other migrated a shorter distance, which made it closer to the negative electrode. As said before, this sample was most similar to the controlled sample B, which would mean the Father was heterozygous, a carrier of sickle cell anemia. Lane 8 was sample F, which was the DNA sample of Child #1. It had only one thing DNA fragment, and it migrated a short distance, which made it closer to the negative electrode side. This sample is most similar to controlled sample A, which would mean this child was homozygous for HbS, making it affected by sickle cell anemia. Lane 9 was sample G, which was Child #2’s DNA sample. It had one thick DNA fragment, which migrated a longer distance, making if closer to the positive electrode side. Compared to the three controlled samples, this sample of DNA was most similar to sample C, which meant this child was homozygous for HbA, making it unaffected by sickle cell disease.

Figure 3

DNA Samples Photographed Under Blue Light

Figure 1 shows the migration of each sample of DNA from the well plates, how many bars each sample had, and which ones matched the original known samples.

Discussion

The results obtained did not match the hypothesis. The genotypes for the father and mother were Rr because they both were heterozygous for sickle cell anemia. The genotype for child one was rr, which meant he/she was affected by sickle cell anemia. The genotype for child two was RR, which meant he/she was not affected by, nor was a carrier of sickle cell anemia. There was a 25% chance for both Child #1 and Child #2’s outcomes. These samples were distinguishable by being homozygous dominant or recessive, or heterozygous because they were compared to the controlled variables of the given DNA samples. About 8% of African children were born as carriers of sickle cell anemia, and about 0.3% were born affected with sickle cell disease (4). Scientists have been working on cures for sickle cell anemia, and have been finding out how to diagnose and see the disease earlier on to the affected person to make it easier to treat (8). Sickle cell anemia affects Even though this disease can be disabling, being a carrier has its advantages, although parents would much rather raise a healthy child than have the chance of giving it this disease (1).

References

(1) Serjeant, G. R., & Serjeant, B. E. (1992). Sickle cell disease (Vol. 3). New York: Oxford university press.

(2) Allison, L. A., Black, M., Freeman, S., Podgorski, G., Quillin, K., Taylor, E. (2016). Biological Science (Vol 6). Pearson, 84-85

(3) Li, S., Sun, B., & SpringerLink (Online service). (2011). Advances in cell mechanics. Berlin, Heidelberg: Springer Berlin Heidelberg. 99-102

 (4) Allison, A. C. (1954). Protection Afforded by Sickle-cell Trait Against Subtertian Malarial Infection. British Medical Journal, 1(4857), 290–294.

(5) Biardi, L., Church, G., Fernandez, A., Phelan, S. (2017). BI 170 General Biology Laboratory Manual. Fairfield, CT: Fairfield University, 53-60

(6) Omoto, C. K., & Lurquin, P. F. (2004). Genes and DNA: A Beginner's Guide to Genetics and Its Applications. New York: Columbia University Press.

(7) Sickle cell disease - Genetics Home Reference. (2012, August).

(8) Hanna, J., Wernig, M., Markoulaki, S., Sun, C. W., Meissner, A., Cassady, J. P., ... & Jaenisch, R. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 318(5858), 1920-1923.

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