A Forensic DNA Microarray Simulation: Detection of Hb Alleles in
Transmission of Plasmodium vivax – Malaria.
By Sanford M.Herzon - 2009, DNA Resource Center, Montgomery County Public Schools.
Objective:
How is DNA technology used to screen for genetic traits & diseases?
Introduction:
There are more than 4,000 genetic diseases currently identified - most are very rare, but some
are relatively widespread, especially within certain ethnic groups. In addition, genetic
predispositions toward conditions such as high cholesterol, heart disease, and cancer have
been found. Most genetic diseases are caused by mutation in the DNA sequence that code for
a malfunctioning protein molecule..
The Genetics:
Sickle cell anemia is a recessive disorder which causes changes in shape of the hemoglobin
protein to become sickle-shaped. This conformation change in the protein molecule causes the
red blood cells in sickle cell patients to become elongated into a crescent shape. This can cause
the red blood cells to become stuck in capillaries (figure 1 on page 4), thus depriving the body
of oxygen and causing all kinds of problems throughout the body such as periodic painful
attacks. Eventually the condition leads to damage of some internal organs, stroke, anemia,
and early death.
People with sickle cell anemia have inherited that abnormal hemoglobin allele, HbS, from
both parents. The normal allele (HbA) codes for normal shaped hemoglobin A person who
receives the defective allele from both father and mother develops the disease (HbS,HbS); a
person who receives one defective and one healthy allele, HbAHbS remains healthy, but can
pass on the disease and is known as a carrier. If two parents who are carriers have a child,
there is a 25% chance of their child developing the illness and a 50% chance of their child
being a carrier (figure 2 on page 4).
Interestingly, it should be noted that the carriers of the sickle-cell trait are almost exclusively
of African, Mediterranean, Indian and the Middle Eastern descent and seem to have an
increased resistance to full malaria. Patients still exhibit symptoms of the illness, but not as
intense. Since malaria is widespread in many parts of Africa, this is a striking example of
evolutionary adaptation in humans where approximately 1 of 13 Africans carries the mutated
allele.
Sickle cell anemia is caused by a mutation in the β-globin (beta) protein chain of hemoglobin.
Hemoglobin contains two protein chains- an alpha chain and a beta chain. The beta chain
contains 287 amino acids. In sickle cell anemia, a point mutation in the DNA gene sequence
has occurred. This is also known as a single nucleotide polymorphism.
TASK: Locate the point mutation:
Normal triplet sequence ______________
Normal Amino Acid ________________
Mutant triple sequence ______________
Mutant Amino Acid ________________
Normal - HbA
Mutant – HbS

The Book of Man, Walter Bodmer and Robin McKie
Normal Hemoglobin
Disease Connection: Sickle cell is not associated with Malaria by chance. These two
diseases have basically evolved together. Malaria kills over one million people, mostly
children, each year. In part of the malaria lifecycle (Figure 3) a mosquito that is infected
with the parasite feeds on a human for blood, the parasite is transmitted into the human host.
Once inside the body, red blood cells become infected and explode releasing more of the
parasites. This causes the classic symptoms of malaria, such as high fever, headaches,
intense chills and sweating. Left untreated, the end result is always death. Cases of malaria
in the United States are very rare, but there have been isolated outbreaks.
The Technology:
What does a microarray or gene chip look like? Most of them look very much like a specimen
slide with a special coating (polylysine) that holds onto the DNA probes.
These probes are assembled one base at a time by a robotic device. An individual’s genetic
code (single strand of DNA or RNA) has a fluorescent color attached to it and is applied to the
probes. Only the complete complementary sequences between the genetic sample and the
DNA probe will bind.
Microarray technology is based on three fundamental principles of DNA:
a. complementary binding of nucleotides: A-T, C-G
b. DNA double helix can be separated into single strands (as in the beginning stages of
replication)
c. Complementary RNA or cRNA can be changed to single stranded DNA – called
cDNA (as in HIV, an RNA virus, making DNA to infect the cell’s DNA)
Figure 1 – Sickle Cell Anemia
Figure 2 – Pattern of Inheritance
National Heart Lung & Blood Institute
Figure 3 – Malaria Life Cycle
CDC Alert – Urgent Message – Centers for Disease Control and Prevention
Local Transmission of
Plasmodium vivax Malaria
--- Palm Beach County, Fl,
2003 -- DNA Testing to
Confirm Genotypes of
Individuals to Begin at
Forensic DNA Lab in
Maryland.
The majority of malaria cases diagnosed in the United States are imported, usually by
persons who travel to countries where malaria is endemic. However, small outbreaks of
locally acquired mosquito-transmitted malaria continue to occur . Despite certification of
malaria eradication in the United States in 1970 , 11 outbreaks involving 20 cases of probable
locally acquired mosquito-transmitted malaria have been reported to CDC since 1992 ,
including two reported in July 1996 from Palm Beach County, Florida (Palm Beach County
Health Department, unpublished data, 1998). This report describes the investigation of four
cases of locally acquired Plasmodium vivax malaria that occurred in Palm Beach County
during July--August 2003. In addition to considering malaria in the differential diagnosis for
febrile patients with a history of travel to malarious areas, health-care providers also should
consider malaria as a possible cause of fever among patients who have not traveled but are
experiencing alternating fevers, rigors, and sweats with no obvious cause. (edited from the
CDC; http://www.cdc.gov)
Case Reports – 2003-001, 002, 003, 004
Case 001. On July 22, a man aged 46 years reported to the emergency department (ED) of
hospital A with a 3-day history of fever, headache, chills, anorexia, nausea, vomiting,
dehydration, and malaise. He was treated with intravenous fluids and discharged with
levofloxacin. On July 24, he returned to the ED with worsening symptoms and was admitted
with a diagnosis of pneumonia. On July 25, P. vivax was identified on a blood smear. The
patient recovered after treatment with doxycycline, quinine, and primaquine. The patient is a
construction worker who reported working outside.
Case 002. On August 19, a man aged 45 years visited the ED of hospital A with a 2-day
history of fever, chills, anorexia, arthralgias, and diarrhea and was discharged on ibuprofen.
The patient visited the ED again on August 21 for these same symptoms, was evaluated, and
discharged. On August 22, he returned to the ED with worsening symptoms and mental
confusion and was admitted; a blood smear demonstrated the presence of P. vivax. He
recovered after treatment with chloroquine and primaquine. The patient slept in a homeless
camp in a wooded area near a canal. He reported using insect repellent.
Case 003. On August 15, a man aged 32 years was admitted to hospital A with a 33-day
history of a mild fever, intermittent chills, headache, nausea, and intermittent sweating. He
had consulted several physicians for his symptoms and had been treated unsuccessfully with
azithromycin and prednisone. On the day of admission, P. vivax was identified on a blood
smear. The patient recovered after treatment with doxycycline, quinine, and primaquine. He
reported having played golf and tennis in the evenings.
Case 004. On August 25, a person aged 17 years was admitted to hospital B with an 8-day
history of a low fever and mild headaches. On August 26, P. vivax was identified on a blood
smear. He recovered after treatment with doxycycline, quinine, and primaquine. The patient
is a student and reported spending time at a pond near his house. .
Epidemiologic Investigation -All four patients reported having no previous history of
malaria, recent blood transfusion, organ transplantation, or intravenous drug use. Three of
the four reported never having traveled to regions where malaria is endemic. All four patients
live within the West Palm Beach area within 10 miles of Palm Beach International Airport.
No international seaport exists nearby. Patients 1 and 2 attended the same local party on
July 4. None of the other patients had any known common activities or interactions.
Laboratory Investigation -Blood specimens were reviewed, and P. vivax infection was
confirmed by both microscopic diagnosis and polymerase chain reaction rRNA gene analysis.
In addition, parasite multilocus genotyping confirmed that all four patients were infected by
the same strain of P. vivax.
Mandated Investigation - DNA samples have been sent to the Forensic Science DNA Labs
to begin genotype screening of the four individuals. Microarray analysis will confirm
genotypes and help design new drug treatments and prevention methods.
Investigators ____________________________ __________________________ Due Date_______
Materials:
boiling water bath
reaction tubes with patient’s blood samples:
1,2,3,and 4
reaction tube for + control, red - HbA
reaction tube for + control, blue - HbS
reaction tube with – control (no color)
probe solution with mixed single stranded
DNA in white staining jars.
plastic droppers (caution: do not cross
contaminate!)
“microarray” slide
slide developing jar
forceps
paper towels
colored pencils (red, blue, purple)
Procedure:
1. Place a copy of the array Figure 1 lab surface.
2. Place a clean slide on top of array Figure 1.
Figure 1
1
2
+R
-C
3
+B
4
Microarray
Slide
2. Add a drop of each liquid solution onto its respective well on the microarray slide.
Key:
1,2,3,4
patient’s blood’s RNA that has been converted to single stranded cDNA.
+R
has known single stranded HbA DNA and should show red once the test is completed. (+
Red Control)
+B
has known single stranded HbS DNA and should show blue once the test is completed. (+
Blue Control)
-C
has no DNA and is thus, should show no color when the test is completed and serve as
the control for the entire procedure.
(- Control)
3. Allow the slide to dry. It should take about 3-5 minutes for the solution to solidify
4. Using forceps, lower the slide completely into the jar containing the HbA and HbS probe
solution.
5. Remove the slide when you notice a color change. This color change should be immediate.
6. Using tweezers place the slide flat on a paper towel to air dry and record the data (colors of
each well)
Data: Well Color of Microarray experiment
Well
Color
Genotype
Person 1 – Case 001
Person 2 – Case 002
Person 3 – Case 003
Person 4 – Case 004
+R
-C
+B
Control
Control
Control
Diagnosis
(Phenotype)
Control
Control
Control
Analysis Questions:
1. Color the spots on your array as shown by your results:
1
2
+R
3
-C
4
+B
2. What does each color indicate?
a. RED – ________________________________________________________________
b. BLUE – _______________________________________________________________
c. PURPLE- _____________________________________________________________
3. What purpose do the controls serve? __________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
4. Evaluate your investigative team’s laboratory skills in this experiment?
_____________________________________________________________________________
_____________________________________________________________________________
5. If these cases should go to court, how well do you think your results would hold up to
the Frye Standard? Daubert Standard?
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________