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The Mystery of the Pnemonia Outbreak

Tuesday 10:10am
Paul Griffith
Paul Adeyemi
Michelle Farrar
Hannah Kozsuch
Fall 2009
 DNA restriction analysis has various benefits in relation to the field of science

and medicine. In order to fully comprehend these benefits we must first understand the

process. Restriction endonucleases function as enzymes that digest Deoxyribonucleic

acid by breaking phosphodiester bonds that link bases together (Grantham, 2009). The

break in sequence of DNA that results is analyzed using the process of gel

electrophoresis. In gel electrophoresis different fragments are separated from one another

as DNA fragments from a restriction digest are pulled through a gel composed of

agarose. This occurs in response to the force of an electrical field. DNA generally travels

from the negative electrode to the positive electrode, moving at a constant rate inversely

proportionally to its physical size. Therefore to visualize these fragments of DNA, we

stain it with EtBr and observe the results under ultraviolet light (Grantham, 2009).

Now that we have briefly explained a portion of DNA restriction analysis, we can

now observe the scientific and medical benefits of the analysis. Scientists have conducted

a study on DNA restriction analysis that entailed the utilization of rhodium (II) acetate as

a restriction enzyme and PCU19 as the DNA (Rahman et al., 2007). Upon the completion

of the experiment, the scientist acknowledged that the restriction enzyme rhodium (II)

acetate has the ability to inhibit DNA replication. They applied this result to note that

subsequent inhibition of cell growth, hence leading to possible mutations. The beneficial

process of DNA restriction analysis helped the scientist to identify the plasmid, thus,

account for possible growth factors that impact organisms.

In another study, scientists utilized DNA restriction analysis to determine the

identification of the Campylobacter (Owens et al., 1989). Similar steps that we conducted

to determine the identity of our mystery plasmid were also performed in their experiment.
The study acknowledged that DNA restriction analysis is beneficial to the field of science

because it provides an accurate process to determine the identity of a plasmid that often

avoids the incorporation of irrelevant information. Therefore, restriction digestions are

often used in science as part of recombinant DNA technology and genetic modification

because of this accuracy.

In this experiment, we were given a mystery strain of bacteria responsible for the

pneumonia outbreak in a South American town. We were given a selection of the

following enzymes to cut the DNA: Acc I, Apa I, Bgl I, Eco RV, Pvu II, and Xmn I

(Grantham, 2009), but we only used two enzymes. Those two enzymes are Bgl I and Apa

I. We chose these two enzymes because we observed from the three strains given that the

Blg I enzyme cuts three times in Strain A and twice in Strain B and Strain C, and Apa I

cuts only once in Strain B. By using these two enzymes, we will know directly which

strain of bacteria is responsible for the pneumonia outbreak.

We used the molecular weight lane as our DNA size standard. The molecular

weight markers depicted how differently each enzyme cut the DNA thus it was essential

in determining the identity of our mystery plasmid. From the picture we obtained from

our experiment, we identified the molecular weight lane as the primary column of DNA

fragments to the left. Upon analyzing the experiment, we used the molecular weight lane

as a point of reference to compare and contrast the different enzymes.

The molecular weight lane assisted us in constructing a semi-log graph. The distance of

each fragment from the base line was measured. We calculated distances of 20mm,

23mm, 26mm, 30mm, 37mm, and 39mm. Upon measuring the distances migrated from
the base line, we plotted the values obtained on the graph in relation to the relative

number of base pairs. A best-fit line was then drawn on the graph.

Lane 1: Standard Base Pairs

Distance from Start Point (mm) Base Pair Size Log of Base Pair Size
20 23130 4.364175633
23 9416 3.97386645
26 6557 3.816705184
30 4361 3.639586087
37 2322 3.365862215
39 2037 3.308991029
Table 1.1 lists the distance from the start point relative to the base pair size and the log of
base pair size.

Standard Curve of distance Traveled by Plasmid

Fragments vs. Base Pair Size

100000

10000
Base Pair Size

1000
Series1
100

10

1
0 10 20 30 40 50
Distance From Start Point (mm)

Graph 1.1 provides the standard curve of distance traveled by the plasmid fragments relative to the
base pairs.
 Standard Curve of Distance Traveled by Plasmid
Fragments vs. Log of Base Pair Size

4
Log of Base Pairs

3 Series1

2 y = -0.0504x + 5.215 Linear (Series1)

R2 = 0.9402
1

0
0 10 20 30 40 50
Distance From Start Point (mm)

Graph 1.2 provides the standard curve of distance traveled by the plasmid fragments relative to the
Log of the base pairs and a standard best fit line with the slope equation. This is used to determine
where the values for the Bgl I and Apa I are relative to the MW line.

Several controls were utilized for our mystery plasmid digestion. These controls

include the plasmid DNA, specific 10X buffers, enzyme, and water. Five micro-liters of

plasmid DNA were inserted into each tube, along with two micro-liters of each specific

enzyme buffer (Grantham, 2009). The difference between these controls and our

molecular weight lane were that for our lane, no enzyme was added to the tube and we

used a random enzyme buffer. In contrast to the other twelve micro-liters of water that we

inserted in all the other tubes (excluding the ladder), we added thirteen micro-liters of

water to the negative control.

 Reaction Plasmid 10X Buffer Enzyme 1 Enzyme 2 H2O Total Volume
Blg I 5 μL 2 μL 1 μL 0 12 μL 20 μL
Blg I + Apa I 5 μL 2 μL 1 μL 1 μL 11 μL 20 μL
Negative 5 μL 2 μL 0 0 13 μL 20 μL
Table 1.2 shows the sample gels prepared to test which strain of bacteria is the correct pneumonia
causing strain.

Strain C through through a process of elimination. We used the graphs that were

made to find the corresponding base pair in relation to the distance migrated for each

enzyme. We used this information to compare fragment sizes from the table to the three

restriction Strains provided in our lab manual. For Strain C, Apa I served as a noncutter.

When we tested for Bgl I we saw that the DNA was cut twice, eliminating Strain A,

which would have been cut three times. In our second sample lane, we tested for Blg I

and Apa I. Since we saw only two cuts and not three, we know that Strain C is the correct

strain of bacteria causing the pneumonia. Strain B would have been cut three times with

the Bgl I and Apa I enzymes. The observed fragment sizes from the table and the photo

from gel electrophoresis support our results as well.

Distance migrated; Observed Fragment Sizes

Unknown plasmid Distance migrated Observed Fragment Sizes

Blg I 35mm 2824.8799749

38mm 1994.3436742
Blg I +Apa I 35mm 2824.8799749
38mm 1994.3436742
Negative Control 32mm 4001.2897354
Table 1.3 lists the distance migrated and the corresponding observed fragment size for the unknown
plasmid.
Image of Gel. Lanes 5, 6, and 7 on top row were used for this experiment.
 Works Cited

Grantham, Charles. Laboratory Manual For Principals of Biology I. Georgia: Edwards

Brothers, Inc ., 2009.

Owens, RJ., Costas, M., & Dawson, C. (1989). Application of different chromosomal

DNA restriction digest fingerprints to specific and subspecific identification of

Camplylobacter isolates. Journal of Clinical Microbiology, 27(10), 2338-2343.

Rahman, MM., Yasuda, H., Katsura, S., & Mizuno, A. (2007). Inhibition of

endonucleases cleavage and DNA replication of E. Coli plasmid by antitumor

rhodium(II) complex. Archieves of Biochemistry and Biophysics. 464(1), 28-35.