RFLP analysis and agarose gel electrophoresis
Purpose
In this lab you will be examining the safety concerns in one type of very commonly used
molecular biology procedure: agarose gel electrophoresis. You will run a mock DNA analysis
called a “restriction fragment length polymorphism” or “RFLP” analysis. This type of
experiment requires the pouring, running and staining of agarose gels. You will
 Use Safety Data Sheets to investigate the chemical hazards associated with agarose gel
electrophoresis

Identify the various hazards associated with pouring, running, staining, and viewing
agarose gels

Learn how to prepare agarose gels

Learn how to safely work with ethidium bromide solutions

Understand the basics of RFLP analysis
Background
Restriction Fragment Length Polymorphism (RFLP) analysis
RFLP analysis is a way to differentiate between the DNA of various species or between
individuals of the same species. Essentially, an RFLP analysis involves cutting up the genome
(which is all the DNA present in a single cell of a particular organism) into pieces of various
sizes that can be separated on an agarose gel. The pattern of sizes that is visualized on the gel
will be a pattern that is unique to that individual or species. (See Figure 1.)
The uses of RFLP analysis are numerous. They include identification tests in criminal cases and
studies on how similar organisms are to one another at the DNA level. In this lab, we will use a
mock RFLP analysis to differentiate between two types of viruses.
1
Figure 1:
A
B
C
DNA
Cut DNA into pieces
C
A
B
Separate by size on agarose gel
Well of DNA gel
Fragment
B
Fragment
A
Fragment
C
2
The major steps in RFLP analysis
DNA from an organism is isolated and cut into pieces with an enzyme called a restriction
enzyme. The pieces will vary in number and size with species or individual. Here, a restriction
enzyme has cut the genome into fragments A, B, and C, which vary in size. These fragments can
be separated by size on an agarose gel, in which the largest fragment (B), runs slowest, and the
smallest (C) runs the fastest. The pattern of fragments viewed on the gel will help with
identification of species or individual.
Agarose gel electrophoresis basics
Agarose gel electrophoresis is one of the most commonly used techniques in molecular biology.
This procedure is a method to separate DNA molecules based upon their size.
Agarose is a polysaccharide that may be dissolved in a buffer containing Tris, Boric Acid and
EDTA. This buffer is called “TBE”. Upon heating of the agarose/TBE mixture, the agarose
dissolves. This mixture is then poured into a casting tray. Upon cooling, the agarose solidifies
into a gel matrix that will act as a sieve for the DNA molecules.
DNA is added to the gel matrix by using a micropipet to load a DNA/ loading dye mixture into
depressions in the gel, called wells. The agarose gel is run in an electrophoresis tank that is filled
with TBE buffer. An electrical current is applied across the gel. DNA is highly negatively
charged, and will, therefore, run towards the positive electrode. As the DNA molecules pass
through the agarose matrix, the molecules separate based, primarily, upon their size. The
smallest DNA molecules traverse the gel the most quickly, while the largest molecules have a
more difficult time passing through the gel matrix.
In order to visualize the DNA molecules within the gel matrix, the gel is stained with a dye
called ethidium bromide. This dye fluoresces under ultraviolet light. When the gel is placed in an
ethidium bromide solution, the dye binds to the DNA in the gel. When the stained gel is placed
on a transilluminator, the DNA molecules will fluoresce.
The mock experimental scenario
In March 2003, a 35 year-old man was diagnosed with “Fifth Disease” (which is caused by a
parvovirus) at a hospital in Albuquerque, NM. He was sent home to recover, but later that same
evening, his symptoms rapidly worsened and he returned to the hospital emergency room. He
was admitted to the hospital, where he received ten days of care until he was well enough to be
sent home to finish his recovery. Over the course of the next two months, 3000 similar cases
were diagnosed in the United States, resulting in 152 deaths.
Although the initial symptoms of this illness very closely mimicked that of the virus that causes
Fifth Disease, a common childhood illness that has few complications, the rapid progression and
high death rate indicated that this was not actually a commonly found parvovirus. The Centers
for Disease Control and Prevention became involved very early in the development of this
potential epidemic. Scientists were able to isolate the virus from patients. They compared the
genome of the parvovirus (causing Fifth Disease) to that of this potentially new and lethal virus
using RFLP analysis. They found that they could rapidly differentiate between the Fifth Disease
virus and this new strain of virus using this technology.
3
Laboratory activity
Overview:
You are working in a diagnostic laboratory. You are ready to run a gel on a previously prepared
RFLP analysis. You are given the following samples:





Sample 1:
Sample 2:
Sample 3:
Sample 4:
Sample 5:
Control RFLP analysis from Fifth Disease virus
Control RFLP analysis from “new” virus
RFLP analysis from patient 1 w/ Fifth Disease symptoms
RFLP analysis from patient 2 w/ Fifth Disease symptoms
RFLP analysis from patient 3 w/ Fifth Disease symptoms
Your goal is to use agarose gel electrophoresis to compare the RFLP results from these three
patients to determine which, if any, of the three are likely to develop the new, potentially fatal
illness rather than show the normal progression of Fifth Disease.
This lab will consist of the following parts:
 Investigation of Safety Data Sheets
 Preparation of an agarose gel
 Loading of the gel
 Electrophoresis of the gel
 Staining of the gel with ethidium bromide
 Photography of the stained gel
 Analysis of RFLP data and diagnosis
Experimental protocol
PART ONE: Investigation of Safety Data Sheets
Examine the Safety Data Sheets for the following chemicals and record any precautions to be
taken in your lab notebook: Tris EDTA
Boric Acid
Ethidium bromide
PART TWO: Preparation of a 1% agarose gel
1. Write out the procedure to make a 1% (w/v) agarose gel in TBE. You will need 30 mL of
agarose for your gel.
2. Weigh out the amount of agarose you calculated in step 1 and place it in a 100 mL flask.
3. Add 30 mL of TBE to the flask.
4. Microwave the flask containing the TBE/agarose mixture for about 45 seconds. WATCH
THE FLASK WHILE MICROWAVING SO THAT IT DOES NOT BOIL OVER.
5. Remove the flask from the microwave USING HEAT-RESISTANT GLOVES and gently
swirl the flask. If you can still see agarose particles, return the flask to the microwave for
about 15 more seconds.
6. Allow the agarose solution to cool to about 50 degrees Celsius.
7. Pour cooled agarose into casting tray with comb as demonstrated by your instructor.
8. Allow gel to solidify.
4
PART THREE: Loading and electrophoresis of the agarose gel
1. Loading dye has been added to your DNA samples. This dye will allow visualization of
how far the DNA has run on the gel and will help the DNA sink into the wells.
2. Remove the comb from the solidified gel.
3. Place the gel, in its casting tray, into the electrophoresis tank. Make sure you put down
the gates of the casting tray.
4. Add TBE to the tank so that the surface of the gel is just covered.
5. Load your DNA samples into the wells of the gel as demonstrated by your instructor.
RECORD WHICH SAMPLE IS LOADED INTO EACH LANE IN YOUR
LABORATORY NOTEBOOK.
6. Connect the leads of the electrophoresis apparatus to the power supply.
7. Run the gel at 120 V for about 40 minutes.
PART FOUR: Staining and photographing the agarose gel
1. Turn off the power supply.
2. Disconnect the leads and remove the casting tray and gel from the electrophoresis tank.
3. Put on gloves.
4. Slide the gel from the casting tray into a Tupperware container set aside for ethidium
bromide use. DO NOT PLACE THE CASTING TRAY OR ANYTHING ELSE DOWN
ON THE BENCH RESERVED FOR ETHIDIUM BROMIDE USE. ALSO, DO NOT
TOUCH THE CASTING TRAY TO THE ETHIDIUM BROMIDE STAINING TRAY.
5. Add ethidium bromide staining solution to just cover the gel in the tray.
6. Stain for 10 minutes with gentle agitation.
7. Using a funnel, carefully pour the ethidium bromide solution back into the stock bottle.
8. Using a spatula, remove the gel from the staining tray and place it on a UV light box.
9. Photograph the picture as demonstrated by your instructor.
10. Discard your gel in the appropriate waste beaker.
Results:
1. Label the lanes of your picture.
2. Compare the results of patients 1 through 3 with the two control samples.
3. Record in your notebook which of the patients is likely to be infected with which virus.
Discussion questions
1. From your study of the Safety Data Sheets, what did you discover about the risk
associated with the use of ethidium bromide?
2. What risks are associated with preparation of agarose gel? What precautions help to
minimize these risks?
3. What risks are associated with running the agarose gel? What precautions help to
minimize these risks?
4. What risks are associated with viewing of the ethidium bromide-stained gel? What
precautions help to minimize these risks?
5. List all special precautions used when working with ethidium bromide.
5