1

DNA Restriction Analysis

Kevin Curran
Honors Biology
Period 3
5/20/16

Introduction
Enzymes have a multitude of functions throughout the world of biotechnology. Each
enzyme has its own very specific functions. Some of these functions include cutting DNA
molecules into pieces. These types of enzymes are called restriction enzymes. Restriction
enzymes themselves are specific to their functions. Each of them cut DNA molecules at a very
specific place. For the restriction enzyme to be able to cut the DNA, the nucleotide sequence
needs to follow the exact sequence that corresponds with the restriction enzyme. Due to these
properties, DNA can be manipulated in ways that can beneficial, such as inserting certain genes
into an organisms genome. This is done by using DNA molecules known as plasmids. These
molecules are small circular DNA that only contain a few genes. 2 This makes it easier to be able
to cut out the unwanted genes, making room for the genes that will result in the desired outcome.
This can be used to aid those that are ailed with genetic disorders. However, inserting plasmids
into our cells is impractical for multiple reasons. For the plasmid to have any large scale effect
on someones health, the plasmids would need to reach every single one of our cells. With the
average human body containing about 37.2 trillion cells, it makes it near impossible for this to
happen.3 The membrane encompassing each of our cells poses another problem. The plasmid
would be unable to pass through the membrane without outside intervention. These problems
lead to looking for organisms where plasmids can easily be implemented into their cells,
bacteria. All bacteria have a special property where they naturally share DNA. This means that
when a plasmid is introduced to a colony of bacteria, many of them will accept it into
themselves, integrating it into their genome. From here, the altered plasmid can produce the
desired outcome.

Another example of the restriction enzymes usefulness is in the process known as RFLP,
the process of mapping out the genome of the organism tested.1 This creates what is referred to
as the organisms DNA Fingerprint. This is done by using a technique called gel
electrophoresis. The purpose of gel electrophoresis is to measure the different lengths of the
DNA fragments after it is cut up by the restriction enzymes. The result is different banded
patterns for each restriction enzyme that is used. This is because after the cut up DNA is placed
into the gel, an electric current is passed through it, causing the DNA fragments to travel through
the gel. The smaller fragments are able to move farther through the gel than the larger
fragments.4 The gel is then stained so that the microscopic DNA can be seen. Each pattern, using
multiple restriction enzymes, is specific to the organism where the DNA was taken. This is why
it is often used in forensics cases to identify criminals. Any DNA left at a crime scene can be put
through gel electrophoresis, where the persons DNA fingerprint is identified.
In our experiment, we used restriction enzymes to demonstrate how gel electrophoresis
works. We used three different restriction enzymes, HindIII, EcoRI, and BamHI, on lambda
DNA. As a control, the lambda DNA was placed into water. If the lambda DNA is mixed with
the three restriction enzymes, then the EcoRI would result in the smallest strands of DNA.
Materials

Four 1.5 mL tubes

Agarose

Centrifuge

Micropipette

37C water

Tris-borate-EDTA buffer

Gel-casting tray

1 L of loading dye

Well-forming comb

Power supply

Procedures
Procedure A: Set up Restriction Digest
1. Label Four 1.5 mL tubes, in which you will perform restriction reactions: B for BamHI, E
for EcoRI, H for HindII, and for no enzyme.
2. Pool and mix reagents by tapping the tube bottom on the lab bench, or with a short pulse
in a microcentrifuge.
3. Incubate all reaction tubes for a minimum of 20 minutes at 37C. You may incubate for
longer.
Procedure B: Cast Agarose Gel
1. Seal ends of gel-casting tray with tape, and insert well-forming comb. Place gel-casting
tray out of the way on lab bench, so that agarose poured in the next step can set
undisturbed.
2. Carefully pour enough agarose solution into casting tray to fill to depth of about 5mm.
Gel should cover only about the height of the comb teeth. Use a pipet tip or a toothpick
to move large bubble or solid debris to sides or end of tray, while gel is still liquid.
3. Gel will become cloudy as it solidifies (about 10 min). Do not move or jar casting tray
while agarose is solidifying.
4. When agarose has set, unseal ends of casting tray. Place tray on platform of gel box, so
that comb is at negative (black) end.
5. Fill box with tris-borate-EDTA (TBE) buffer, to level that just covers entire surface of
gel.
6. Gently remove comb, taking care not to rip wells

7. Make certain that sample wells left by comb are completely submerged. If dimples are
noticed around the walls, slowly add buffer until they disappear.
8. The gel is now ready to load with DNA. If you will be loading the gel during another
period, you need to cover the electrophoresis tank to prevent dying of the gel.
Procedure C: Load Gel
1. Add 1 L loading dye to each reaction tube. Mix dye with digested DNA by tapping tube
on lab bench, or with a pulse in microcentrifuge.
2. Use micropipette to load contents of each reaction tube into a separate well in gel, aligned
as illustrated in Ideal Restriction Digest of lambda DNA. Use fresh tip for each reaction
tube.
a. Steady pipet over well using two hands
b. Be careful to expel any air in micropipette tip before loading gel. (If air bubble
forms cap over bell, DNA/loading dye will flow into buffer around well edges.)
c. Dip pipet tip through surface of buffer, position it over the well, and slowly expel
the mixture. Sucrose in the loading dye weighs down the sample, causing it to
sink to the bottom of the well. Be careful not to punch tip of pipet through bottom
of gel.
Procedure D: Electrophoresis
1. Close top of electrophoresis chamber and connect electrical leads to an approved power
supple, anode to anode (red to red) and cathode to cathode (black to black). Make sure
both electrodes are connected to same channel of power supply.

2. Turn power supply on and set voltage as directed by your instructor. Shortly after current
is applied, loading dye can be seen moving through gel toward positive pole of
electrophoresis apparatus.
3. The loading dye will eventually resolve into two bands of color. The faster-moving,
purplish band is the dye bromophenol blue; the slower-moving, aqua band is xylene
cyanol. Bromophenol blue migrates through gel at the same rate as DNA fragments about
300 base pairs long. Xylene cyanol migrates through gel at a rate equivalent to about
2000 base pairs.
4. Allow the DNA to electrophorese until the bromophenol blue band nears the end of the
gel.
5. Turn off power supply, disconnect leads from inputs, and remove top of electrophoresis
chamber.
6. Carefully remove casting tray and slide gel into staining tray.
Results

This is a picture of what the ideal results of this

experiment would have been.

log(Kbp)

HindIII
Distance log(Kbp)
37
1.439
1.600
42
1.364
55
0.974
1.400
65
0.817
80
0.640
1.200
111
0.366
120
0.307
1.000

log(Kbp) vs. Distance (mm)

0.800

0.600
0.400
0.200
0.000

10

20

30

40

50

60

70

80

90

100

110

120

130

140

Equation
Distance (mm)
-.0133129108(x) + 1.813797785

y = -0.0133x + 1.8138

This graph was created using the measured distances of HindII from the picture of the ideal gel along
with the actual number of base pairs that corresponded with each distance

Using the measurements gathered from the picture of the ideal gel and the given number
of base pairs that goes with each of the distances, an equation to calculate the number of base
pairs given the distance was created. This can be used to find the number of base pairs for the
other restriction enzymes. The results can be seen in the chart below.
HindII

EcoRI

BanHI

Dis.

Act. Bp.

Dis

Cal. Bp.

Act. Bp.

Dis.

Cal. Bp.

Act. Bp.

37

27,491

40

19,111

21,726

45

16,395

16,841

42

23,130

60

10,352

7,421

50

14,065

12,275

55

9,416

70

7,619

5,643

60

10,352

7,233

65

6,557

75

6,536

4,878

65

8,880

6,770

80

4,361

90

4,127

3,530

70

7,619

5,626

111

2,322

120

2,027

This table compares the actual number of base pairs to the ones that were calculated

Discussion
After concluding the experiment, I am able to see that my hypothesis was false. The
restriction enzyme that produced the shortest DNA fragments was the HindII enzyme. However,
I was able to make the conclusion that the different restriction enzymes do cut the DNA in
different places. I was also able to determine that the smaller fragments were able to travel much
farther through the gel than the longer fragments. There are multiple places during the
experiment where there could have been errors that resulted in our own gel not showing any of
the bands. A few of them include sucking the DNA back into the pipet when attempting to insert
the DNA into the wells and not running the electrophoresis long enough for the DNA and dyes to
travel through the gel. Another thing that could have gone wrong was that the gels were stained
for too long.

References
"Introduction." NCBI. U.S. National Library of Medicine, n.d. Web. 17 May 2016.1
"Mama Ji's Molecular Kitchen." ASU - Ask A Biologist. 12 Apr 2010. ASU - Ask A Biologist,
Web. 18 May 2016.2
Eveleth, Rose. "There Are 37.2 Trillion Cells in Your Body." Smithsonian. N.p., 24 Oct. 2013.
Web. 18 May 2016.3
Nature.com. Nature Publishing Group, n.d. Web. 19 May 2016.4