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Topic/Case- A patient is
suspected to have sickle
cell anemia. Describe, how
you could possibly
diagnose the disease using...
• autosomal, genetic blood disorder
• decreased number of Red blood cells ( RBCs)
• the protein hemoglobin is affected
• t...
• Painful swelling of the hands and feet,
known as dactylitis
• Fatigue or fussiness from anemia
• A yellowish color of th...
 caused by point mutation in the B-globin chain
 at 6th position the hydrophilic Glutamic acid gets replaced
with the hy...
 the sickle cell gene is passed from generation to
generation in a pattern of inheritance called
autosomal recessive inhe...
 the nucleotide sequence coding for the 4-7 residues are different
for the normal hemoglobin chain (HbA) and the sickled
...
• the normal chain has three restriction sites
whereas the defective chain has only two
restriction sites.
 the restricti...
 separates fragments of DNA, RNA and even proteins
based on their molecular weight
 process requires a well of gel where...
 1. Dissolve the agar, cool the solution, and pour the gel. Combine agar and water.
Bring the mixture to a boil and heat ...
 5. Place the lid on the chamber and connect the electrode leads to the power
supply. Connect the black lead to the negat...
 Gel electrophoresis can be used to-
 Solve criminal cases
 Solve paternity cases
 Diagnose genetic disease
 Determin...
 So the steps to test for sickle cell anemia in patient can be –
 Collect the blood sample of the patient. In adults, a ...
 If the patient has the disease, then will
possess the sickle cell DNA, and therefore
the electrophoresis results of the ...
 When hemoglobin from people with severe sickle cell anemia, sickle cell trait, and
normal red blood cells was subjected ...
 If the patient is a carrier and has sickle cell trait, then the hemoglobin
electrophoresis from the patient’s blood samp...
 Screening test
 Newborn screening
 Prenatal screening
 It is better to redo the tests and diagnosis
once the results ...
a) Sickle cell disease is the most common genetic
disorder in the United States.
b) People are born with sickle cell disea...
1. Bone marrow transplant. A bone marrow transplant, also known as a
stem cell transplant, is the only cure for sickle cel...
SICKLE CELL ANEMIA
SICKLE CELL ANEMIA
SICKLE CELL ANEMIA
SICKLE CELL ANEMIA
SICKLE CELL ANEMIA
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SICKLE CELL ANEMIA

How SICKLE CELL ANEMIA possible could diagnose using the gel electrophoresis .

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SICKLE CELL ANEMIA

  1. 1. Topic/Case- A patient is suspected to have sickle cell anemia. Describe, how you could possibly diagnose the disease using Gel Electrophoresis.
  2. 2. • autosomal, genetic blood disorder • decreased number of Red blood cells ( RBCs) • the protein hemoglobin is affected • the RBCs change in shape and become rigid and sticky • survive only for 10-20 days unlike the case of normal RBCs surviving for 90-120 days
  3. 3. • Painful swelling of the hands and feet, known as dactylitis • Fatigue or fussiness from anemia • A yellowish color of the skin, known as jaundice, or whites of the eyes, known as icteris,that occurs when a large number of red cells hemolyze
  4. 4.  caused by point mutation in the B-globin chain  at 6th position the hydrophilic Glutamic acid gets replaced with the hydrophobic valine  sickle hemoglobin sticks together and form long rods
  5. 5.  the sickle cell gene is passed from generation to generation in a pattern of inheritance called autosomal recessive inheritance  both the mother and the father must pass on the defective form of the gene for a child to be affected  if only one parent passes the sickle cell gene to the child, that child will have the sickle cell trait  W  with one normal hemoglobin gene and one defective form of the gene, people with the sickle cell trait make both normal hemoglobin and sickle cell hemoglobin
  6. 6.  the nucleotide sequence coding for the 4-7 residues are different for the normal hemoglobin chain (HbA) and the sickled hemoglobin chain ( HbS).  in case of the defective hemoglobin chain, the allelic sequence is mutated and therefore the B-globin chain loses a restriction site at the position where the glutamic acid is replaced by valine.
  7. 7. • the normal chain has three restriction sites whereas the defective chain has only two restriction sites.  the restriction fragment for the DNA with the mutated allele is larger than the restriction fragment for the DNA with the normal allele  the electrophoretic bands are larger when an individual has two alleles of one type.
  8. 8.  separates fragments of DNA, RNA and even proteins based on their molecular weight  process requires a well of gel where and electric field is introduced which induces the movement of the target molecules based on their charge and size  because DNA and RNA are negatively charged molecules, they will be pulled toward the positively charged end of the gel  bands representing molecules of different sizes can be detected  the bands for the samples can be compared to a ladder in order to detect what type and kind of sample is present
  9. 9.  1. Dissolve the agar, cool the solution, and pour the gel. Combine agar and water. Bring the mixture to a boil and heat until the agar is dissolved. Cool the agar until you can comfortably touch the flask. Place tape across the ends of the gel form (if needed) and place the comb in the form. Pour cooled agar into the form; the bottom 1/3-1/2 of the comb should be immersed. Immediately rinse and fill the agar flask with hot water to dissolve any remaining agar. When the agar has solidified, carefully remove the comb. Remove the tape (if used) from the ends of the gel form.  2. Load samples in the wells in the gel.  3. Place the gel in the electrophoresis chamber with the wells closest to the negative (black) electrode.  4. Prepare the salt solution and add it to the chamber. Add salt to tap water and swirl it to dissolve. Fill each half of the chamber, adding solution until it is close to the top of the gel. Then gently flood the gel from the end opposite the wells to minimize sample diffusion.
  10. 10.  5. Place the lid on the chamber and connect the electrode leads to the power supply. Connect the black lead to the negative terminal and the red lead to the positive terminal.  6. Turn on the power supply and adjust the voltage to 50-100 volts.  7. Run the gel for 5-10 minutes .You will be able to observe the samples separating into different colors. Placing the chamber on white paper will help you see the color separation.  8. Turn off the power supply, disconnect the electrode leads, and remove the chamber lid.  9. Remove the gel from the electrophoresis chamber. You may also remove the gel from the form and place it on a piece of plastic wrap. Placing the gel on a piece of white paper will help you better see the results. Record and evaluate the results of the electrophoresis.  10. Clean up. Discard the gel in the trash and pour the salt solution down the drain. Rinse the electrophoresis chamber and gel form with tap water; turn them upside down to dry
  11. 11.  Gel electrophoresis can be used to-  Solve criminal cases  Solve paternity cases  Diagnose genetic disease  Determine genetic kinship among species
  12. 12.  So the steps to test for sickle cell anemia in patient can be –  Collect the blood sample of the patient. In adults, a blood sample is drawn from a vein in the arm. In young children and babies, the blood sample is usually collected from a finger or heel. The sample is then sent to a laboratory, where it's screened for hemoglobin S.  After storage and proper incubation, the Red blood cells can be harvested.  The DNA of the RBCs can be isolated, by degrading the protein and RNA in the culture with phenol and RNase enzymes and centrifuging the contents to obtain the DNA in the supernatant. This is done by lysing the cells first. The DNA sequences are fragmented with restriction enzymes .  The hemoglobin protein must be extracted from the harvested culture of the red blood cells. This can be done with by lysing the RBCs with a detergent and then treating the lysed contents with DNase and RNase and then centrifuging the culture.  The DNA and protein extract of the patient, who is suspected to have sickle cell anemia should be subjected to gel electrophoresis. The bands should be carefully observed and then results should be recorded.
  13. 13.  If the patient has the disease, then will possess the sickle cell DNA, and therefore the electrophoresis results of the patients DNA sample will show only one single band , as due to mutation, the DNA loses one restriction site at the B-globin chain.  If the patient is healthy and normal, the results will show two bands of DNA, indicating that the patient has a normal B- globin chain.
  14. 14.  When hemoglobin from people with severe sickle cell anemia, sickle cell trait, and normal red blood cells was subjected to electrophoresis, the following interesting results were obtained –
  15. 15.  If the patient is a carrier and has sickle cell trait, then the hemoglobin electrophoresis from the patient’s blood sample will produce two separate bands , each band representing a normal hemoglobin protein and a sickled hemoglobin protein, where the later would be heavier than the normal protein and would migrate to a lesser distance than that off the normal hemoglobin protein in the gel run.  If the patient has sickle cell anemia, then a single band will be produced after electrophoresis. The single band will be at a position which is dissimilar to the position for the normal hemoglobin protein. The proteins will also migrate a lesser distance ( till the middle of the gel ) than the normal proteins do because the sickled hemoglobin are heavier than the normal ones due to the point mutation and alteration of the glutamic acid sequence by valine sequence at the sixth position of the B- globin chain.  If the patient is not diseased and healthy then, a single band will appear showing that the protein has migrated towards the end of the gel.
  16. 16.  Screening test  Newborn screening  Prenatal screening  It is better to redo the tests and diagnosis once the results show an indication of the disease or the trait, so that the patient can be given proper medication and treatment.
  17. 17. a) Sickle cell disease is the most common genetic disorder in the United States. b) People are born with sickle cell disease; it does not develop in adulthood, and it is not contagious. c) Sickle cell disease is chronic but treatable and is not a death sentence. d) Sickle cell disease affects people of many different races. e) Patients with sickle cell disease require comprehensive care.
  18. 18. 1. Bone marrow transplant. A bone marrow transplant, also known as a stem cell transplant, is the only cure for sickle cell anemia. It involves replacing the affected bone marrow with bone marrow donated by someone without sickle cell anemia. After the transplant, the new bone marrow will produce healthy blood cells  All people who have SCD should see their SCD care providers regularly. Regularly means every 3 to 12 months, depending on the person’s age. The SCD doctor or team can help to prevent problems by: • Examining the person • Giving medicines and immunizations • Performing tests • Educating families about the disease and what to watch out for • Antibiotics like penicilln

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