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

1. Topic/Case- A patient is
suspected to have sickle
cell anemia. Describe, how
you could possibly
diagnose the disease using
Gel Electrophoresis.

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

4. • 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

5.  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

6.  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

8.  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.

9. • 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.

10.  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

11.  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.

12.  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

13.  Gel electrophoresis can be used to-
 Solve criminal cases
 Solve paternity cases
 Diagnose genetic disease
 Determine genetic kinship among species

14.  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.

17.  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.

18.  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 –

20.  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.

21.  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.

22. 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.

23. 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