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Biology
LAB: In Search of My Father (Gel Electrophoresis)
Experiment Objective
 Students will learn how agarose gel electrophoresis separates different sizes of dyed molecule that represent
DNA fragments.
 Students will learn how these fragments form unique DNA patterns (“fingerprints”) for each person, which is the
basis for solving maternity and paternity identity.
Introduction
In a war-torn country, two young boys were separated from their respective parents who had been imprisoned
by the regime. The children were cousins who were born two months apart and looked strikingly similar. Their mothers
were half-sisters who shared a common mother (Figure 1). After several years, the regime was overthrown and
replaced by a new government that released all political prisoners from prison. The prisoners were reinstated in their
respective communities and all charges against them were dropped.
During this period, the two boys had not been separated from each other, and had been adopted by a highranking military officer who had no children of this own. Both the officer and his wife died in a liberation uprising and
for a short period of time the boys were placed in an orphanage. On their 18th birthday, the boys were released from
the orphanage and they immediately began to search for information regarding their lost parents.
The brothers contacted a number of elders from their village. The elders informed them that after their
biological parents were released from prison, one of the fathers was assassinated by individuals who were sympathetic
to the fallen regime. The second biological father was found in the village, suffering from amnesia. Two women, who
were thought to be their biological mothers, were in a rehabilitation facility that housed approximately 200 women.
Upon arrival at the center it was very obvious that the patients had not received proper medical attention. Based on age
and appearance, about ten women fit the profile of the two mothers.
Determination of Parentage using DNA Fingerprinting
First, the boys sought to determine which of the ten women were their mothers by performing mitochondrial
DNA fingerprinting. Mitochondria, the “powerhouse” of the cell, are unique organelles in that they contain a small DNA
genome – evidence they may have been a separate organism at some point in our evolutionary history (the
endosymbiotic theory). This genome is useful for identifying maternity because mitochondria are inherited only through
the female line. Before conception, a human egg contains a large number of mitochondria; in contrast, human sperm
contain very few mitochondria. Upon fertilization of the human egg by a sperm, the developing zygote contains
mitochondria obtained from the mother’s egg.
Mitochondrial DNA fingerprinting tests can be used as an initial screening technique because they are less
expensive than chromosomal DNA testing and results are available in a shorter period of time. In this case, since the
boys were cousins (their mothers were half-sisters who shared the same mother), the mitochondrial testing results
would be identical for the two boys. The tests identified two women with mitochondrial DNA fingerprinting patterns
that matched that of the boys.
In order to match the boys with the correct parents, chromosomal DNA fingerprinting tests were ordered for the
boys, the mothers, and the surviving father. Chromosomal DNA, which is present in the nucleus of every living cell, is
the genetic material that acts as a blueprint for all of the proteins synthesized by that cell. Unlike mitochondrial DNA,
chromosomal DNA is an equal combination of both parents. In each chromosome pair, one is inherited from the father
and the second from the mother. Although most of this DNA is identical between individuals (all humans are over 99%
genetically similar), small sequence differences called “polymorphisms” occur at specific locations throughout the
genome. These polymorphisms include single base pair changes in the DNA and repetitive DNA elements. By examining
several of these polymorphic regions, we can generate a unique “DNA fingerprint” for that person.
DNA fingerprints can allow us to distinguish one individual from another. Because polymorphisms are inherited,
DNA fingerprints can also be used to determine paternity, maternity, or other familial relationships. To determine
parentage, the DNA fingerprints of the boys are compared with the DNA fingerprints from the surviving father and the
two mothers. Since chromosomal DNA is inherited from both parents, the DNA fingerprint of a child will contain a
mixture of polymorphisms from each parent. The pattern is easily recognizable as matching visible DNA bands on the
gel and can be experimentally demonstrated in this simulation experiment.
Pre-Lab Questions
1.
What is the difference between mitochondrial DNA and chromosomal DNA?
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2.
Why can mitochondrial DNA only be used to determine maternity (motherhood) and not paternity
(fatherhood)?
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3.
What is a DNA fingerprint? Is it different than a person’s fingerprints they have on their palms?
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4.
Why are polymorphisms useful in creating DNA fingerprints?
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5.
Figure 2 below shows the DNA fingerprints of four different individuals (not related to this scenario).
Position of wells
FIGURE 2: Gel electrophoresis results for a paternity test
With reasoning, explain who fathered the child.
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LAB: In Search of My Father (Gel Electrophoresis)
Experiment Objective
 Students will learn how agarose gel electrophoresis separates different sizes of dyed molecule that represent
DNA fragments.
 Students will learn how these fragments form unique DNA patterns (“fingerprints”) for each person, which is the
basis for solving maternity and paternity identity.
Procedure:
1. Using water, practice the correct use of the micropipette as shown by Mrs. McCarter.
Practice measuring 15 microliters in the micropipette.
2. Place the casting tray/gel in the buffer chamber. You should have enough buffer to cover the
gel. Mrs. McCarter will set this up for you.
3. Load the wells with 15 microliters of each color of dye. #1 on the left and #6 on the right. Use
a new micropipette tip for each color!
Dye Number
1
2
3
4
5
6
Individual
Mother 1
Father
Mother 2
Boy 1
Boy 2
Standard
4. Place the orange lid on the chamber.
5. Plug in the power source, and press the power button to turn it on. DO NOT OPEN THE
CHAMBER OR TOUCH THE GEL WHILE THE POWER SOURCE IS ON!
6. The dyes will separate/move over 10 to 20 minutes.
7. After the dyes have finished, turn off the power source, open the lid, and remove the casting
tray.
8. Remove the gel from the casting tray, and record the results.
9. Carefully clean up all supplies.
Data: Sketch a drawing of your gel in the space below. Include measurements, labels for the wells, and color.
Discussion Questions
1.
Look at samples listed in Table 1. Which sample was the control group in this experiment? How could you tell?
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2.
a) Based on the evidence obtained in this experiment (see your photograph of the gel results), which child,
mother, and father are reunited? (Check the correct boxes)
Mother 1 □
Boy 1 □
Mother 2 □
Boy 2 □
Father □
b) Referring to the gel results, explain why you picked the child, mother, and father you did in part a).
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