The Use of Restriction Fragment Length Polymorphism to Diagnose a Suspected Cystic Fibrosis
Patient
Debra Jones
Abstract:
Restriction fragment length polymorphism was used as a method in order to diagnose
Jeff. Jeff is a young boy who is a suspected Cystic Fibrosis patient. Jeff has been having
symptoms that are congruent with Cystic Fibrosis. RFLP was used. Two different restriction
enzymes were spliced with Jeff’s DNA. The resulting fragments were then run through a gel
with both positive and negative controls. Based on the results of this test, it was determined that
Jeff did have Cystic Fibrosis.
Introduction:
The purpose of this experiment was to diagnose a young boy named Jeff. It was
hypothesized that Jeff has Cystic Fibrosis. Using Restriction fragment length polymorphism,
Jeff’s DNA was tested to determine whether or not he had Cystic Fibrosis. Cystic Fibrosis is a
genetic disease that is caused by a defective gene that affects the lungs and digestive tract. This
gene causes the body to produce thick, stick mucus that obstructs the pancreas and the pathway
of its enzymes. This inhibits digestion. The mucus also blocks the air passage way and causes
lung infections. Symptoms of Cystic Fibrosis include: Wheezing, shortness of breath, frequent
lung infections, a persistent cough, salty skin, weight fluctuation, and greasy stools (Riordan et
al, 1988).
One method that is used to test for cystic fibrosis is called Restriction fragment length
polymorphism. RFLP is a technique that uses restriction enzymes to break apart DNA at their
restriction sites. This causes different lengths of DNA fragments to be made. These fragments
are then run through a gel to determine how many cuts were made by the restriction enzyme
(Nagaoka and Ogihara, 1997). Normal DNA usually has more restriction sites that DNA that
comes from someone with cystic fibrosis. This is due to a mutation in one of the restriction sites
for people that have cystic fibrosis.
Methods:
Restriction digest preparation:
In order to diagnose Jeff, first 10 µL of reaction buffer as added to 15µL of Jeff’s DNA.
This process was repeated for 15µl of both a positive and negative control DNA. Each of these
steps was repeated so that there was two of each of the previously mentioned sample solutions.
Next, 15 µL of enzyme 1 was added to one of the Jeff DNA samples and one of the negative
DNA samples, and 15µL of enzyme 2 was added to the other Jeff DNA and the negative DNA
samples. These enzymes cut the DNA at specific restriction sites. Based on the size of the pieces
of the DNA fragments, one can tell whether Jeff has Cystic Fibrosis or not. The samples were
next incubated for thirty minutes in a 37⁰C water bath.
Gel preparation:
A 0.8% gel was prepared by using agarose TAE buffer, and ethidium bromide. After the
gel was made, 5 µL of loading dye was added to each of the restriction digest solutions. They
were then placed on ice. Next, the samples, positive controls of DNA cut with enzyme 1 and
enzyme 2, and marker DNA were loaded into the well. The gel was then run at 120 V for thirty
minutes. The gel was then viewed and imaged under a UV light.
Results:
The gel shows that Jeff’s DNA that was restricted by both enzyme 1 and enzyme 2
contained two sizes of DNA fragments (Image 1). Wells 4 and 8 each contain Jeff’s DNA, and
they each exhibit 2 bright bands at 2150 bp and 4700 bp for the DNA with enzyme 1, and 1800
bp and 7200 bp for DNA with enzyme 2 (Image 1 and Figure 2). The further down the band is
the larger the DNA fragment is.
Image 1 The gel contains the following in
each well from left to right: well 1- ladder;
well 2- Negative control with enzyme 1; well
3- Positive control with enzyme 1; well 4Jeff’s DNA with enzyme 1; well 5-blank; well
6- negative control with enzyme 2; well 7positive control with enzyme 2; well 8- Jeff’s
DNA with enzyme 2
Standard Curve
Distance in Cm
10
y = 0.9726e0.0002x
Distance
Expon.
(Distance)
1
0
5000
10000
Bp
Figure 1 The standard curve of the
gel. The length of the DNA
fragments in Base pairs was plotted
against the distance the bands
traveled down the gel in cm.
Well
Number
Marker
Negative
Positive
Jeff,
Negative
control,
control,
Enzyme 1 control,
enzyme 1
enzyme 1
4
2
2
1:1500
1: 2150
2:1300
2: 4300
Positive
Jeff,
control,
enzyme 2
enzyme 2
enzyme 2
2
3
2
2
1: 2150
1: 2150
1:1800
1: 1800
1:1800
2:4300
2:4300
2:5800
2:5800
2:5800
of bands
Bp
3: 5000
3: 7200
4: 3000
Figure 2 Includes the size of the DNA
fragments, and how many there were.
The standard deviation of the length of the bands in the gel and the distance that they
traveled was found. It was represented by the equation y = 0.9726e0.0002x. The standard deviation
exhibits a positive curve (Figure 1).
Conclusion:
Based on the data collected from the RFLP gel, Jeff likely has cystic fibrosis. During the
RFLP process, two sets of three types of DNA were loaded into the gel. The first set contained a
positive control, a negative control, and Jeff’s DNA that were spliced with enzyme 1. The second
set contained the same three types of DNA, but these were spliced with enzyme 2. For the DNA
cut with enzyme 2 there are only two restriction sites that are 1800 and 7200 bp in length. A
normal, healthy person would have three restriction sites that would be 1500, 5800, and 1800 bp
in length. This helps support Jeff’s diagnosis. Further support is provided by both the positive
control and Jeff’s DNA having bands that are the same length. The positive control gave an
example of what DNA of someone with cystic fibrosis would look like.
As previously mentioned, the positive control is to provide an example of what DNA that
is affected by cystic fibrosis would look like. For enzyme 2, it contained two fragment sizes of
DNA instead of the normal three. The negative control served as a baseline for the experiment. It
showed what normal, non-cystic fibrosis DNA looked like. For enzyme 2, it showed the normal
three lengths of the DNA fragments that were created by the restriction sites.
Even the slightest change such as a loss of a single amino acid in a protein can cause
devastating effects. This is due to an alteration of the DNA sequence. This change could also
affect the chromosomes, possibly causing them to deleted, duplicated or possibly inverted. The
changes to the chromosomes and the DNA sequencing would cause a change in genetic
expression. Because the DNA template is different, the proteins that are created will also be
different, therefore causing different genetic expression (Clancy, 2008).
This experiment could be furthered by using other methods to test Jeff’s DNA for Cystic
Fibrosis. More data could be collected to support the findings of this experiment.
References:
Clancy S. Genetic Mutation. Nature Education 1(1):187, 2008.
Nagaoka T, Ogihara Y. Applicability of inter-simple sequence repeat polymorphisms in wheat
for use as DNA markers in comparison to RFLP and RAPD markers. Theoretical and Applied
Genetics 94(5): 597-602, 1997.
Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S,
Plavsic N, Chou J, Drumm ML, Iannuzzi MC, Collins FS, Tsui L. Identification of the Cystic
Fibrosis Gene: Cloning and Characterization of Complementary DNA. Science 245: 1066-1073,
1988.