Beverly1
Introduction
From previous research amino sugars have been known to cause virus
inactivation, DNA strand scission, or DNA modification. Many have hypothesized that
the increase of DNA damage or modification brought about by these amino sugars and
their derivatives can lead to mutagenesis, carcinogenesis, and biological aging (Morita
449). Since reducing sugars are present in many biological organisms it is only natural
that their effects should be investigated.
The main focus of my experiment is to investigate the effects of D-Glucosamine
on the E. Coli plasmid pAMP. There were two main factors that were examined in this
experiment. The first was what effects if any does D-Glucosamine have on plasmid
DNA and the second to what degree will it cleave DNA. Gel electrophoresis was used in
order to examine the DNA. The only variable that was examined was time. The
concentration of both the DNA and amino sugar were held constant.
D-Glucosamine
Beverly2
In order to understand how reducing sugars can modify DNA it is first necessary
to explain the different forms that plasmid DNA will take. The first form is closed
compact circular DNA or “supercoiled DNA”. In this form the plasmid has a circular
shape and is tightly wrapped around itself by hydrogen bonds. When the DNA is nicked
by an enzyme of some sort it will change from its ccc-DNA to open circular DNA. DNA
in this form will keep it circular shape but will no longer be wrapped about itself. If the
enzyme has the ability to break both strands of the DNA the will be form linear DNA.
All 3 forms of the DNA will maintain the same molecular weight but the have
now undergone conformational changes. These changes are what will be used in order to
assay the DNA cleavage. The compact shape of the ccc-DNA has the ability to travel
through a gel faster than the other two forms. Linear DNA would move the second
fastest. The slowest band would be the band corresponding to open circular DNA.
Beverly3
The three forms of DNA also have the capacity to bind with one another of the
same form to produce dimers and even multimers. This can easily be seen on a gel as a
large band traveling at a slower rate than the other 3 forms. Dimers and multimers travel
at a rate which corresponds to the rate at which the monomer travels.
1
The amount of DNA was quantified using the Image J program. The program measures
the brightness of a band by calculating the intensity of each pixel.
1
http://rsb.info.nih.gov/ij/
Beverly4
Materials and Methods
Preparation of Solutions for DNA isolation*
GTE Solution
40% sterile glucose
.5 M EDTA pH 8
1 M Tris HCl pH 8
ddH20
2.27 ml
2.00 ml
2.50 ml
93.2 ml
Lysis Solution
1 N NaOH
10% SDS
ddH20
2.00 ml
1.00 ml
7.00 ml
3 M Potassium Acetate
5 M KOAc
Glacial Acetic Acid
ddH20
60.0 ml
11.5 ml
28.5 ml
RNAase 10 mg/ml
Dissolve 100 mg RNAase A in 10ml of 10mM Tris HCl 15mM NaCl. Heat to 100
degrees Celsius for 15 minutes. Cool slowly to room temperature.
*
All solutions must be made under sterile conditions.
Preperation of E. Coli Plasmid pAMP
Inoculate 50 ml of LB broth containing 50 micrograms per ml of ampicillin with a
bacterial colony containing the pAMP plasmid. Incubate overnight at 37 degrees
Celsius. Spin the cells down at 2500 rpm for 15 min and discard the supernatant.
Resuspend the pellet in 2ml of GTE solution and incubate at room temperature for 5
minutes. Add 4 ml of fresh lysis solution mix gently and place on ice for 5 minutes.
Add 3 ml of of ice cold 3M Potassium Acetate and place on ice for 5 minutes.
Centrifuge for 1 minute at 4 degrees Celsius and transfer supernatant to a clean tube
and incubate at room temperature for 2 minutes. Add 60% the volume of isopropanol
Beverly5
to the supernatant and incubate at room temperature for 2 minutes. Pellet the nucleic
acid and discard the supernatant. Wash the pellet twice with 70% ethanol and discard
the supernatant. Dry the pellet and resuspend in 5 ml of TE buffer pH 7.5. Add 10
µL of a 10 mg/ml solution of RNAase and incubate for 30 minutes at room
temperature. Assay the DNA to determine the purity and the amount using UV-Vis
spectra.
260 = 1.03
280 = 0.515
260/280 = 1.99
Find the ratio of the 260/280. This ratio was found to be 1.99. A typical ratio is between
1 and 2. According to literature it is stated that 1 abs unit is equal to 50 µg of DNA. The
amount of DNA isolated was found to be 25.75µg.
Beverly6
Preparation of Amino sugar for DNA Cleavage
First of all a stop solution should be prepared using 22.5 mM EDTA (pH 8.2), 1.5% SDS,
25% (w/v) sucrose, and .02% (w/v) bromophenol blue. The final solution should be
100µL, contain a known concentration DNA, and .1 M D-Glucosamine. Incubate at 37
degrees Celsius over a six hour period. The control consisted of DNA that was unaltered
by the amino sugar but had been incubated for the full six hours. Experimental samples
(10 µL) were removed from incubation at one hour intervals. The stop solution (3 µL)
was added. The sample was then placed in the freezer until assayed.
Preparation of DNA for Gel Electrophoresis and Analysis
Made a 1% agarose gel containing 2 µg per mL of Ethidium Bromide. Ethidium
Bromide was used in order to prevent band smearing and increase resolution. The lane
assignments were as followed. Lane 1 contained the reference DNA which was 1µL of λ
DNA. The rest of the lanes contained 10 µL of the reaction mixture starting with the
control and ending with hour six. The next step was to stain the gel with Ethidium
Bromide for 10 minutes then destain it with water. After the gel has been destained with
water a picture was taken under UV light. The picture should be scanned and saved as a
jpeg image. Image J program was used in order to quantify the data. The first thing that
should be done is to select a lane. When the values were found the information was
saved and transferred over to a spreadsheet. The intensity of each band was then
converted into a percentage and plotted vs. time. The ccc-DNA should be multiplied by
1.4 in order to correct for the Ethidium Bromide absorbance.
Beverly7
Gel ran on February 19, 2004
A solution was made containing 5.15x10-4 µg of DNA, 10µL of 1 M DGlucosamine dissolved in 50mM Tris-HCl buffer pH 7.2 (final concentration .1 M), and
89 µL of Tris-HCL. The incubation period was started at 12:45 PM. The incubation
period lasted from 12:45 PM to 6:55 PM. At the end of the incubation period the samples
were place in the freezer overnight to be run in the morning. A 1 percent agarose gel was
used to run the samples. 10 µL of the DNA solution containing the amino sugar and stop
solution was added to each well. 1µg of λ DNA was added to the first lane as a
reference. The gel was run for 65 min. at 88 volts. The gel was then stained in Ethidium
Bromide for 10 minutes and destained for 10 minutes. The picture was taken and
scanned so that it could be ready for the Image J program.
Gel ran on February 19, 2004
There was not enough DNA present to come to any valid conclusions in this
experiment. There were bands present but it was just that they were so faint that it was
Beverly8
difficult for the software distinguish between the actual bands and background noise.
Even though the picture was taken it was not used in the assessment.
Beverly9
Gel ran on February 24, 2004
A solution was prepared containing 2.575x10-2 µg/µL, 10µL of .1M amino sugar,
and 85 µL of 50 mM Tris-HCl buffer. The incubation period was started at 9:50 am.
The incubation time went from 9:50 am to 3:50 pm. When the allotted time was reached
3 µL of stop solution was added to a 10 µL aliquot. The samples were then place in the
freezer until it was time to run the gel. The samples were run on a 1% agarose gel
containing 2 µg/mL of Ethidium Bromide. The gel was ran for 80 minutes at 93 volts.
Gel Ran February 24, 2004
Control
Hour 0
29
2
8
61
100
Hour 1
28
2
8
62
100
Hour 3
30
2
8
61
100
Hour 4
23
3
17
57
100
Hour 5
14
3
18
65
100
Hour 6
15
4
21
60
100
8Supercoiled
5Nicked
26ccc-Dimer
61Nicked Dimer
100Total
Beverly10
Hour 1(top) vs. Hour 3 (bottom)
By comparing the first hour to hour three it easy to see that there is a difference in
intensity. Each peak corresponds to a different band. The first peak on the left is the
peak that represents the ccc-DNA. This peak is has lowered in intensity as the time
progresses. The area under the first peak for hour one is 30%. The value under the first
peak for hour three is 23%. The values of the ccc-DNA decrease over time. One thing of
interest is that there is a linear band of DNA that has appeared due to degradation of the
plasmid not caused by the reducing sugar. This linear band does not seem to change as
dramatically as the other bands do over time. The control is quite different than the DNA
that was incubated for six hours in the amino sugar. This proves that the DNA is not
affected by its incubation at 37 degrees Celsius
y = -3.5652x + 30.957
R2 = 0.9039
% of DNA Present
35
30
25
20
15
10
5
0
0
1
2
3
4
Time in Hours
5
6
7
Beverly11
Gel ran on February 26, 2004
A solution was made containing 1.545x10-3 µg of DNA, 10 µL of .1M amino
sugar, 87 µL of 50 mM Tris - HCl. The incubation period was started at 9:55 am. The
procedure went from 9:55 am to 4:01 pm. When the right time was reached 3 µL of the
stop solution was added to a 10 µL aliquot of the sample. After incubation the samples
were placed on ice until it was time to run the gel. The samples were run on a 1%
agarose gel containing 2 µg of Ethidium Bromide per 1 mL of .5M Tris-HCl buffer (pH
7.2). The gel was run at 93 volts for 117 minutes. The results were as shown.
Gel ran on February 26, 2004
Control
Hour 0
22
16
36
5
20
100
Hour 1
20
7
15
23
35
100
Hour 3
18
6
20
14
43
100
Hour 4
13
8
25
3
51
100
Hour 5
13
8
32
1
46
100
Hour 6
8
8
32
0
51
100
5Supercoiled
6Linear
35Nicked
0ccc-Dimer
54Nicked Dimer
100Total
Beverly12
One major feature that was noticed is that the fourth band or peak is also fading as
time progresses. This is more than likely due to the fact that this band is a ccc-DNA
dimer or multimer. The control does not seem to be affected by the incubation period so
it is safe to say that incubating DNA at 37 degrees Celsius is not enough to cause any
conformational changes. It seems that the ccc-DNA is being converted to the oc-DNA.
This would be the third band down. When looking at the values it is evident that the
amount of oc-DNA seems to be increasing as time progresses. The interesting thing is
that it does not seem to form the oc-dimer. The value of the oc-dimer increased after the
sugar was added but it did not seem to vary that much afterwards. The linear band is also
present in every sample in this trail. This band behaves in the same way the linear band
did on the gel run February 24, 2004.
y = -2.4161x + 20.484
R2 = 0.962
% of DNA Present
25
20
15
10
5
0
0
1
2
3
4
Time in Hours
5
6
7
Beverly13
Gel Ran on March 4, 2004
A solution was prepared containing 2.575x10-2 µg/µL, 10µL of .1M amino sugar,
and 85 µL of 50 mM Tris-HCl buffer. The incubation period was started at 8:45 AM.
The incubation lasted from 8:45AM to 2:55 PM. Once the hour of interest was reached
10 µL of the DNA solution was added to 3 µL of stop solution and then placed in the
freezer until it was time to run the samples on a gel. A 1% agarose gel was made in the
absence of ethidium bromide. 10 µL of the sample was then added to its corresponding
well. λ DNA was added to the first well as a reference.
Gel ran on March 4, 2004
Lane 2
Lane 3
23
8
22
13
34
100
Lane 4
19
7
18
18
38
100
Lane 5
20
7
21
15
38
100
Lane 6
15
7
28
7
44
100
Lane 7
13
7
29
4
47
100
Lane 8
9
8
34
2
47
100
9Supercoiled
7Linear
33Nicked
1ccc-Dimer
50Nicked Dimer
100Total
Beverly14
Control (top) vs. Hour 6 (bottom)
The ccc-DNA in this experiment followed the same pattern as in the other
experiments. It decreased over time and almost completely disappeared by hour six. The
same behavior is seen in the ccc-DNA multimer. As shown in the graph by hour six the
peak had completely vanished. This is evidence that D-Glucosamine has the ability to
nick plasmid DNA. The linear peak is also present in this gel as well. This is just due to
the plasmid being stored for long periods of time. When the DNA was incubated over
the six hours the linear band did not show any evidence of being cleaved by DGlucosamine.
y = -1.9814x + 20.441
R2 = 0.9336
% of DNA present
25
20
15
10
5
0
0
1
2
3
4
Time in Hours
5
6
7
Beverly15
Conclusion
After running the trials it was easy to see that D-Glucosamine does have the
ability to nick plasmid DNA. From the experiments that were performed it is difficult to
tell the specificity. What is known is that ccc-DNA is nicked because the amount of cccDNA decreases as it is incubated with the reducing sugar. There was not much evidence
suggesting that the DNA was transformed into oc-DNA. Two out of the three trials that
were studied showed this trend but there was not enough information to prove that this
was normal behavior caused by D-Glucosamine.
One problem that was encountered was the degradation of the DNA plasmid. It
was difficult to tell if the D-Glucosamine was actually cleaving the plasmid enough to
form linear DNA. This experiment showed that linear DNA was not obtained from the
amino sugar reaction. Even though great care was taken in order to prevent the DNA
being cleaved by outside factors it still happened. Even though the formation of linear
DNA was not intentional it helped to prove that D-Glucosamine does not have the ability
to cleave linear DNA into smaller fragments.
This experiment gave enough evidence to show that D-Glucosamine does have
the ability to nick ccc-DNA. The experiment also showed that the amino sugar had no
affect on linear DNA. This was shown using a crude method of DNA quantification
validity of this statement comes into question. The problem is that it could not prove
much more than that. More tests would have to be run in order to make more
conclusions.
Beverly16
There are many other opportunities that can be explored using this same
methodology. This experiment just looked at time as a variable. Other experiments can
be set up using amino sugar concentration or pH as a variable. Another type of amino
sugar could be used to see how well that cleaves the DNA. Instead of just concentrating
on the types of sugars that cleave DNA a procedure could be set forth in order to see what
types of compounds could be used in order to inhibit the cleavage of DNA. As stated
previously this research could be important because amino sugars are present throughout
all biological organisms.
Beverly17
Bibliography
1. T. Komano and J. Morita, Agric. Biol. Chem., 47: 11-18 (1983).
2. C. Helms, http://hdklab.wustl.edu/lab_manual/plasmid/ptsmid05.html. (1990).
3. J. Sagripanti and S. Toyokuni. Journal of Inorganic Biochemistry, 47: 241-248
(1992)
4. S. Fujii, S. Nanjou, K. Tanaka, K. Ueda, and T. Komano, Agric. Biol. Chem, 48:
1865-2867 (1984).
5. P. Sauer, M. Muller, and J. Kang, Qiagen GmbH, 2: 23-269 (1998).
6. K. Watanabe, N. Kashige, Y Nakashima, M. Hayashida, and K. Sumoto, Agric. Biol.
Chem., 50: 1459-1465 (1986).
7. J. Morita, K. Ueda, S Nanjo, and T. Komano, Nucleic Acids Research, 13: 449-458
(1985).