Lab Report N*9 Sickle-Cell Anemia PDF

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Laboratory reports for Spring 2018 Cell & Molecular Biology Laboratory by Professor. Henri P. Antikainen and Cervantes. ...

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Molecular Biology of Sickle-Cell Anemia Introduction: In today’s experiment we will use agarose gel to separate two allelic forms of the redblood-cells in hemoglobin, responsible for oxygen exchange in higher animals. By doing so, we will investigate that sickle-cell anemia is caused by a point mutation at the position 6 of Hemoglobin subunit b. Hemoglobin is a tetramer composed of two different types of polypeptides known as alpha and beta, hence, Hb = alpha (subscript 2) + beta (subscript 2). In blood the most common form is Hemoglobin A (HbA) known as the normal form. Changing a base of the nucleotide in the gene for the beta subunit causes the substitution of an amino acid to residue creating a negatively charged lateral group. Mutations causes the nucleotides to change bases from an A substituted to a U, therefore, changing the protein as well. The point mutation being investigated here is a substitution or nucleotides where an A nucleotide in the triplet code protein of Glutamic acid was substituted by uracil: GAA  GAU. This changed of nucleotide changed the whole amino acid frim Glutamic acid to Valine, which has a nonpolar lateral group. The allelic form HbS is found in people with sickle cell disease and sickle cell traits. When having sickle-cell traits, the hemoglobin tetramers are mutated and hence the molecules are altered. This makes the structure of the cell fragile and causes them to break more easily when going through capillaries, causing sickle-cell disease, known as falciform anemia. To determine the disease’s presence, if and only, one allele of the mutated gene is present the person is heterozygote for the disease. If HbA is produced predominantly, then a new condition is found called sickle-cell trait, which does not produce occlusion of blood vessels and does not adversely affect the individual’s life expectancy. Objective: We will separate the two allelic forms of hemoglobin: the normal molecule, HbA, and HbS by electrophoresis in agarose gel. Methods: 1. Gel preparation: use tris-glycine buffer without its denaturing agent SDS 2. Preparation of Femoglobin samples with the given blood samples: a. Normal individual b. Sickle-cell trait c. Sickle-cell anemia 3. Electrophoresis of the gel Preparation of solutions  a. 25 mM tris-100 Glycine, of pH 9.4 b. 3 g of Tris base c. 7.2 g Glycine d. Dissolve in 900 mL distilled water

e. Adjust volume to 1 L with distilled water Agarose Gel  a. Weight 1.t g of powdered agarose and add 100 ml of the buffer b. Swirl to mix and microwave for 3 minutes c. Cool down the solution Hemoglobin A 10 mg/ml  5mg H-A in .5 mL distilled water Hemoglobin S 10 mg/ml  5mg H-A in .5 mL distilled water Loading buffer  5ml Glycerol, 5 mL distilled water, dispense in ten 1-mL aliquots and keep frozen at -20*C until used Data: This photograph represents our Agarose gel containing HbS and HbA to determine the individuals containing normal, sickle-cell trait, and sickle-cell anemia blood sample (from left to right).

Discussion-Questionnaire: In this experiment, we investigated the use of agarose gel to separate two allelic forms of the red-blood-cells in hemoglobin. We found that a point mutation took place at the position 6 of the subunit b Hemoglobin, which change the nucleotides of the protein Glutamic acid. The change of nucleotide, known as substitution changed the nucleotide sequences from GAA to GAU, therefore, affecting its protein and structure. As the nucleotides were change, the protein changed from Glutamic acid to Valine, of a nonpolar lateral group. As the molecules are altered, we expect to see changes in the structure making it fragile and easy to break when undergoing capillaries, which would therefore cause the sickle-cell disease. In order to determine the disease’s presence, if and only, one allele of the mutated gene is present the person is heterozygote for the disease. If HbA is produced predominantly, then a new condition is found called sickle-cell trait, which does not produce occlusion of blood vessels and does not adversely affect the individual’s life expectancy.

1. The agarose gel we obtained during this experiment came out to be as the expected migration in the gel. HbS or HbA alone, would therefore, appear alone, HbA in the form of a normal individual’s blood sample, while HbS, containing sickle-cell anemia. In the other hand, when both appear together, the sickle-cell trait was found.

2. The theoretical values for HbA and HbS’ molecular weight comes out to be HbA = 64.4 kDa HbS = 64.3 kDa 3. Yes, the change of Glu to Val protein explains the unequal migration of HbA and HbS in my gel at the DNA level, as the mutation lead to a conformational change in the DNA. Normal cell tends to move faster in the agarose gels than sickle-cell trait or sickle-cell anemia. Also, when HbS and HbA are together, there is a thicker migration. 4. Yes

5. Another disease associated with the estraneous forms of Hemoglobin subunits in the human adult is HbE, known as Hemoglobin E which is cause by Glu26Lys mutation in the beta-globin. Having two HbE would cause a mild anemia to occur. 6. The type of point mutation in which AU change occurs in the codon for the sixth amino acid in hemoglobin chain B, is called Transition. 7. The term heterozygotes advantage means that the genotype has a higher fitness than either homozygous dominant and/or homozygous recessive genotype. Traits or genotypes in advantage are likely to be shown/pass down to the next generation through natural selection in animals or reproduction in humans. 8. The advantage of people having sickle-cell traits in regions where malaria is a major cause of death is that sickle-cell traits in HbA and HbS can act as a resistance to malaria. This resistance would therefore allow the probability of those individuals to live a longer life’s expectancy. Conclusion: Finally, in this experiment, being able to separate the two allelic forms of hemoglobin: the normal molecule, HbA, and HbS by electrophoresis in agarose gel, allowed us to understand the principles and signs of sickle-cell diseases. Although sickle-cell diseases could potentially be deathly, we learned how they work as a defense system to malaria. Overall, the agarose gel we obtained was the expected and correct gel, meaning no error or complications were found when completing our experiment. Bibliography: Luzzatto, L. (2012). Sickle Cell Anaemia and Malaria. Mediterranean Journal of Hematology and Infectious Diseases, 4(1), e2012065. http://doi.org/10.4084/MJHID.2012.065. Published 2012 October 3 by NCBI https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499995/....