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DNA

RESTRICTIO
N ANALYSIS
Marissa Beck
Honors Biology
May 12, 2016
Pd. 3

Introduction:
Usually found in bacteria, restriction enzymes are used to cut specific fragments of a
DNA sequence. Restriction enzymes are sequence specific and they recognize specific DNA
sequences (1). For example, the restriction enzyme BamHI attaches to the DNA sequence
GGATCC. The restriction enzyme EcoRI cuts DNA at the recognition site GAATTC and HindIII
cuts DNA at AAGCTT. All three of the aforementioned restriction enzymes work best at thirtyseven degrees Celsius (3). A DNA sequence informs scientists the genetic information that is
carried in a specific DNA segment. In the DNA double helix, the four chemical bases always
bond with the same partner to form base pairs. Adenine permanently pairs with thymine and
cytosine always pairs with guanine. These pairs are the basis for the way DNA molecules are
copied and are crucial for cell division (2).
Scientists use restriction enzymes to cut DNA into smaller fragments (1). Gene
technology is the term that includes certain actions that concern understanding gene expression,
taking advantage of natural genetic variation, modifying genes, and transferring genes to new
hosts (4). A genome is an organisms complete set of instructions. The simple steps of
recombinant DNA is to take out and cut up DNA from a donor genome into fragments from one
to multiple genes. Also, it allows these fragments to insert themselves one at a time into freely
replicating DNA molecules, known as vectors (5). Today, DNA is used for criminal and paternity
cases. Different methods, such as RFLP, are used to determine these cases. RFLP stands for
Restriction Fragment Length Polymorphism Technique and is used by comparing the DNA
from a suspect to a piece of DNA from the crime scene. This technique has become increasingly
popular for criminal and paternity cases (6).

Gel electrophoresis is a method that sorts DNA, RNA, or proteins according to their
molecular size. Gel electrophoresis is used in order to help scientists or researchers better
analyze DNA according to their size. Gel electrophoresis works because charged molecules
move through the gel when an electric current is passed across it. This shows the points that
restriction enzymes cut the DNA. In order to see the DNA fragments, scientists use dyes to give
color to sample, give weight to sample so it would sink into well, and to show the progress of gel
migration (7). Gel Electrophoresis works because DNA is negatively charged because of all its
phosphate groups in the backbone of the DNA. As a result, DNA is attracted to the positive
charge. As the pieces of DNA move through the gel, they will feel resistance. Larger pieces of
DNA will move at a slower rate than smaller fragments of DNA (8).
The purpose of this lab was to examine if different restriction enzymes cut DNA into
different sized fragment. Also, another purpose of this lab was to introduce restriction enzymes
and gel electrophoresis. Finally, we wanted to create a logarithmic graph with known data to
determine the other lengths of the DNA fragments that were created by the other restriction
enzymes cutting them. The independent variable of this lab were the distances the DNA
fragments traveled and the dependent variable were the DNA fingerprints, or the banded patterns
on the gel. The control group of this experiment was the test tube without a restriction enzyme,
just water. My hypothesis for this experiment was: If I have four test tubes with lambda DNA
with three different restriction enzymes BamHi, EcoRi, and HindIII, and one with water, then I
will end up with different sized DNA fragments.

Materials:

DNA and Restriction Enzymes


o BamHi
o EcoRI
o HindIII
Water
Lambda DNA
TBE Buffer
Loading Dye
Staining Solution
Agarose
Eight-Well Comb
Microcentrifuge Tubes
Staining Trays
Disposable Gloves
Dry Labs
Micropipets
Pipets
Gel Chamber
Water Bath at thirty-seven degrees Celsius
Ultra-violet Light
Power Supply
Millimeter Ruler

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 HindIII, and for no enzyme.
2. Use table below as a checklist while adding reagents to each reaction. Read down each
column, adding the same reagent to all appropriate tubes; use a fresh tip for each reagent.
All groups share the same BamHI, EcoRI, HindIII enzymes at a central station.
Tube

DNA

Buffer

BamHI

EcoRI

HindIII

H2O

B
4 L
5 L
1 L
E
4 L
5 L
1 L
H
4 L
5 L
1 L
4 L
5 L
1 L
3. Pool and mix reagents by tapping the tube bottom on lab bench, or with a shirt pulse in
microcentrifuge.
4. Incubate all reaction tubes for a minimum of twenty minutes at 37C. Your teacher may
instruct you to incubate the reactions for a longer period.

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 next step can set undisturbed.
2. Carefully pour enough agarose solution into casting tray to fill to depth of about 5 mm.
Gel should cover only about 1/3 the height of comb teeth. Use a pipet tip or toothpick to
move large bubbles 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 a 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 wells, 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, your teacher will instruct you to cover the electrophoresis tank to prevent drying
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 micropipet load contents of each reaction tube into a separate well in gel, aligned as
illustrated in Ideal Restriction Digest of Lambda DNA. Use a fresh tip for each reaction
tube.
a. Steady pipet over well using two hands

b. Be careful to expel any air in micropipet end before loading gel. (If air bubbles
forms cap over well, DNA/loading dye will flow into buffer around edges of
well.)
c. Dip pipet tip through surface of buffer, position it over 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: Electrophorese
1. Close top of electrophoresis chamber and connect electrical leads to an approved power
supply, anode to anode (red-red) and cathode (black-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 same rate as a DNA fragment
approximately 300 base pairs long. Xylene cyanol migrates at a rate equivalent to
approximately 2,000 base pairs.
4. Allow the DNA to electrophorese until the bromophenol blue band nears the end of the
gel. Your instructor may monitor the progress of electrophoresis in your absence; in that
case, omit steps 5 and 6.
5. Turn off power supply, disconnect leads from the inputs, and remove top of
electrophoresis chamber.
6. Carefully remove casting tray and slide gel into staining tray labeled with your group
name. Take gel to your instructor for straining.
7. Examine your stained gel on a light box or overhead projector. Compare your gel with the
ideal gel shown, and try to account for the fragments of lambda (9).

Results:

Picture of Tested Restriction


Digest of Lambda DNA

Picture of Ideal Restriction


Digest of Lambda DNA

FIGURE 1: This picture above depicts the end


result of my tested gel No bands or DNA
fingerprints appeared on this gel.

FIGURE 2: This picture above shows what the


gel should look like after the experiment. This
ideal gel clearly shows the DNA fingerprints.

As seen in Figure 1, the tested gel, no bands or DNA fingerprints appeared. In Figure 2,
the ideal gel given in the lab manual, all three restriction enzymes showed the DNA fingerprints.
All of the distances throughout the Results were taken from the Ideal Gel. I used a millimeter
ruler to measure the distance between the gel well at the top of the picture to the top of each
DNA fingerprint for each restriction enzyme. Some of the DNA fingerprints were measured at
both the top and bottom.

FIGURE 3: This graph shows the HindIIIs points of distance in millimeters and its log kilo base pairs.
Included in this graph is HindIIIs line of best fit and its equation.

Size of Base Pairs vs. Distance Travled


1.6
1.4
1.2

f(x) = - 0.01x + 1.82

Log (kbp)

HindIII

0.8

Linear (HindIII)

0.6

Linear (HindIII)

0.4
0.2
0
30

40

50

60

70

80

90 100 110 120 130

Distance (mm)

In Figure 3, the graph depicts the size of base pairs of the restriction enzyme HindIII
compared to the distance it traveled through the gel based on the ideal gel in Figure 2. The
distance was found in millimeters and the base pairs are the log of the kilo base pairs. After
plotting the points on the graph, I found the line of best fit, which also gave me the equation of
the line. The equation for this graphs line of best fit is y = -0.0134x + 1.8196.

Distance Traveled and Calculated Base Pairs for Restriction Enzymes


HindIII
Distance Act. Bp.
(mm)
(kbp)
37
*27.491
42
*23.130
56
9.416
65
6.557
80
4.361
111
2.322
120
2.027
**0.564
**0.125

Distance
(mm)
40
45
60
67.5
72.5
75
90

EcoRI
Cal. Bp.
(kbp)
19.213
16.466
10.366
8.224
7.049
6.525
4.108

Act. Bp.
(kbp)
*24.756
*21.226
7.421
*5.804
*5.643
4.878
3.530

Distance
(mm)
45
50
65
67.5
70

BamHI
Cal. Bp.
(kbp)
16.466
14.112
8.884
8.224
7.614

Act. Bp.
(kbp)
16.841
12.275
7.233
6.770
5.626

*Pair appears as single band. **Does not appear on this gel


FIGURE 4: This table shows the distance traveled for each restriction enzyme from the ideal gel. The actual base
pairs were given, but the calculated base pairs were solved for. The distance in this table is in millimeters and the
base pairs are in kilo base pairs.

In the chart above, the restriction enzymes are listed with their distance traveled from the
gel well, their calculated base pairs and their actual base pairs. The HindIII restriction enzymes
actual base pairs were given. I used that information to make a graph (Figure 3) and find its line
of best fit equation. Once the equation was found, I inputted the distance for the x-value. The
output of this equation was the calculated kilo base pairs. I repeated this equation for each
distance that I found for the EcoRI and BamHI. After finding all of the calculated kilo base pairs,
I compared the number with the actual kilo base pairs given by my teacher.

Discussion:
The restriction enzymes did create different sized DNA fragments, proving my
hypothesis correct. The restriction enzyme BamHI cut the lambda DNA forty-five, fifty, sixtyfive, sixty-seven and a half, and seventy millimeters from the gel well. EcoRI split apart the
DNA forty, sixty, seventy, seventy-five, and ninety millimeters away from the gel well. Finally,
the restriction enzyme HindIII cut the DNA at thirty-seven, forty-two, fifty-six, sixty-five, eighty,
one hundred and eleven, and one hundred twenty millimeters from the gel well. This proves that
restriction enzymes do cut DNA at different locations. The smaller sized fragments traveled
farther through the gel because they migrated through the gel more quickly than larger-sized
fragments that migrated more slowly and therefore had travelled a shorter distance.
There were many sources of error that could have happened through the entire process of
the lab. For the actual lab portion, we did not use a micropipet or an ultraviolet light due to the
minimal resources we had. Instead of using a micropipette, we opted to use a flimsy, plastic
pipet. While inserting the DNA into the gel wells, we might have sucked in some of the solution
or may have broken the wells. We also might have not even inserted the DNA into the wells
properly. Another source of error was the amount of time the gel electrophoresis ran. Instead of
letting the electrical current run through the gel for an hour and a half like the lab manual
suggested, we only ran it for about twenty minutes. Also, the DNA might have been stained for
too long and we did not use an ultraviolet light.

References:
(1) ""DNA Restriction" Biology Animation Library." DNALC.org. Cold Spring Harbor
Laboratory, n.d. Web. 15 May 2016.
(2) "DNA Sequencing Fact Sheet." Genome.gov. National Human Genome Research Institute,
18 Dec. 2015. Web. 15 May 2016.
(3) Thermofisher.com. Thermo Fisher Scientific Inc., 2016. Web. 15 May 2016.
(4) "Overview of Gene Technology Research at CSIRO." CSIRO.au. Commonwealth Scientific
and Industrial Research Organization, n.d. Web. 15 May 2016.
(5) Griffiths, Anthony JF. "Making Recombinant DNA." Ncbi.nlm.nih.gov. U.S. National
Library of Medicine, 2000. Web. 15 May 2016.
(6) "DNA Evidence in Criminal Cases." Nolo.com. Nolo, 2016. Web. 15 May 2016.
(7) "DNA Gel Electrophoresis." The Dictionary of Genomics, Transcriptomics and Proteomics
(2015): 1. 29 Nov. 2005. Web. 16 May 2016.
(8) Yip, Alice. "How Does Gel Electrophoresis Work?" Futurescienceleaders.org. Budding
Scientists, 20 Mar. 2013. Web. 16 May 2016.
(9) Micklos, David A., and Greg A. Freyer. DNA Restriction Analysis Kit. DNA Science: A First
Course in Recombinant DNA Technology. Cold Spring Harbor Laboratory and Carolina
Biological Supply Company, 1995. Print.