Peter Touresian
BCH 467 Lab
Thursday; 12:00pm
Dayn Sommer
11 September 2014
Vector Identification and Restriction Mapping Through Restriction Endonuclease
Digestion and Agarose Gel Electrophoresis
Abstract
The objective of this experiment is to identify whether the unknown vector obtained in
the given plasmid is pRSETB or pQE30, by the construction of a restriction map and an
observation of specific restriction sites by restriction endonuclease cleavage. The initial Plasmid
obtained, Plasmid A, was digested and fragmented by three known restriction endonucleases,
with identities of BamHI, PstI, and ScaI. Single digestions of each restriction endonuclease were
used along with double digestions with different combinations of these enzymes. The several
DNA samples with the different enzymes were electrophoresed in an agarose gel electrophoresis
apparatus, which displayed DNA fragments that could be measured to approximate the size of
each fragment. Analysis of the data ascertained by the many fragments seen on the gel was used
to map the specific restriction sites of each endonuclease. The obtained Plasmid A’s identity was
the vector, pRSETB. This vector contained 4 restriction sites with a calculated size of 3.9 kb
pairs.
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Introduction
The main objective of this experiment was to determine the DNA map of an unknown
Plasmid A by the determination of restriction sites where three distinct restriction endonucleases
(enzymes) cut the DNA. Once the enzymes digest the DNA on the DNA strand specific to the
restriction endonuclease, the distance between adjacent cuts are calculated to properly map the
plasmid and to determine whether the unknown vector is pRSETB or pQE30. The restriction
enzymes and the DNA plasmid were incubated in an environment for the enzymes to be
optimally active and then placed in an agarose gel electrophoresis apparatus to determine the
migration distances of the several DNA strands that were cut by the restriction enzymes.
A plasmid is an extra-chromosomal circular strand of DNA comprised of four kilo base
pairs that undergoes replication independently from the host chromosome1. Plasmids are entirely
found in the cytosol of bacteria, yeast, and other fungi and incorporate specialized sequences that
enable them to utilize the cell’s resources for their own replication and expression of genes1.
Naturally occurring plasmids usually have a symbiotic role in the cell1. They may provide the
genes that allow resistances to antibiotics or provide the ability to perform new functions for the
cell1. Some plasmids carry the gene for the 𝛽-lactamase enzyme, which allows for the resistance
to 𝛽-lactam antibiotics such as penicillin, ampicillin, and amoxicillin1. Plasmids may be
transferred from bacterial cells of the same or different species that are antibiotic-resistant to
ones that are antibiotic-sensitive yielding the new recipient, antibiotic-resistant, in an exclusive
process known as conjugation1.
Restriction endonucleases are enzymes that recognize certain DNA sequences, which
lead to the plasmid to be digested (cut) at that specific site. The majority of the specific sites for
these enzymes are palindromic sequences, where both DNA strands are the same and
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complimentary to each other when read from their respective 5’ to 3’ end. Three different types
of restriction endonucleases exist and are denoted by types: I, II, and III.1 Type I restriction
endonucleases cleave DNA at random sites that range up to one thousand base pairs passed the
recognition site.1 Type III restriction endonucleases cleave DNA up to twenty-five base pairs
passed the recognition site1. Both of these types require the energy stored in ATP to be powered1.
Type II restriction endonucleases are the most abundant, with thousands that are known1. This
type requires no ATP hydrolysis and is known as the simplest of the three types.1 The
mechanism by which restriction endonucleases cut their specific DNA sequences is through the
hydrolysis of the phosphodiester bond on both sides of the DNA. In this experiment, the use of
three type II restriction endonucleases were implemented. The identity of these enzymes are:
PstI, ScaI, and BamHI. PstI has a restriction site of cleavage for the sequence: 5’-CTGCAG2.
ScaI has a restriction site of cleavage for the sequence: 5’-AGTACT2. BamHI has a restriction
site of cleavage for the sequence: 5’-GGATCC2.
Both the plasmid and the restriction endonucleases were placed in an incubator, which
allows for the most optimal environment of activity for the mechanisms of the restriction
endonucleases to take place. Following, a laboratory technique used to separate the different
DNA fragments of plasmid; known as agarose gel electrophoresis was implemented. Agarose
gels are generally used for DNA samples that are larger in size ranging from seventy base pairs
to eight hundred kilo base pairs3. In agarose gel electrophoresis, DNA migrates from the
negatively charged cathode to the positively charged anode with the induction of an electric
field, due to DNA’s exhibition of a negative charge3. The mobility of DNA from the well, down
the agarose gel is inversely correlated to the fragment size3. The smaller the DNA fragment, the
farther down its travel3. Conversely, the larger the DNA fragment, the shorter the travel3. This
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travel distance is described as the migration distance and measured in centimeters. Therefore,
when plotted on a graph, the migration distance is directly proportional to the log of the size of
the DNA fragment. More specifically, in this experiment the equation for the graph (seen on
Graph 1) was: log (kb pairs) = (-0.3115 * migration distance) + 1.616.
Materials and Methods
Seven micro-centrifuge tubes were used. The first three were single digestions of Plasmid
A with three different restriction endonucleases. The following three were double digestions of
Plasmid A with different combinations of restriction endonucleases. The last tube was purely
Plasmid A without any restriction endonucleases resulting in uncut DNA. The first three tubes
contained 10 µL of 1X digestion buffer and 5 µL of 50 ng/µL Plasmid A. Following, 5 µL of
BamHI (2 U/µL) was added to the first tube, 5 µL of PstI (2 U/µL) was added to the second tube,
and 5 µL of ScaI (2 U/µL) was added to the third tube. The following three tubes (tubes four
through six) consisted of double digests. These three tubes contained 5 µL of 1X Digestion
buffer and 5 µL of 50 ng/µL Plasmid A. In the fourth tube, 5 µL of BamHI (2 U/µL) and 5 µL of
PstI (2 U/µL) were added. In the fifth tube, 5 µL of BamHI (2 U/µL) and 5 µL of ScaI (2 U/µL)
were added. In the sixth tube, 5 µL of PstI (2 U/µL) and 5 µL of ScaI (2 U/µL) were added. As
mentioned, tube 7 contained no restriction endonucleases, just 15 µL of 1X digestion buffer and
5 µL of 50 ng/µL Plasmid A. Amongst all of the seven tubes, a total volume of 20 µL was
reached. All of these tubes were then placed in an incubator at 37oC for 45 minutes.
As the incubation process was proceeding, the agarose gel was prepared. The gel tray was
filled with molten agarose and SYBRsafe. Over the course of the incubation process the prepared
gel was set aside to solidify at room temperature. Once solidified, the gel was placed in the
electrophoresis apparatus and 300mL of 0.5X TBE was added to the gel and apparatus. Once the
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incubation was complete, 5 µL of loading buffer was added to each tube and centrifuged.
Following, 20 µL of each sample was placed in a well on the gel. In the remaining eighth well, a
10 µL, one kilo base ladder size marker was added. The gel was then put under electrophoresis
for an hour at a constant 100 volts from the power supply. In about an hour, the dyes had
migrated about 2/3 of the way, which was necessary to end the electrophoresis. After the
electrophoresis, a Kodak digital gel imaging system photographed the gel.
Results
Figure 1
Plasmid A
1. BamHI
2. PstI
3. ScaI
4. BamHI + PstI
5. BamHI + ScaI
6. PstI + ScaI
7. Uncut
8. DNA Ladder
Figure 1is the photo image of
the agarose gel produced by the Kodak digital gel-imaging system of the DNA digests with the
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use of BamHI, ScaI, and PstI. The first three wells (from left to right) were the result of a single
digest. The following three were the result of a double digest of a different combination of 2
enzymes. The seventh well was the uncut DNA fragment, and the eighth well was the 1 kb DNA
ladder that was used as a guide to determine the fragment sizes of the migration distances of each
band. The DNA samples in each well ran from the cathode (top of gel) to the anode (bottom of
gel). Due to only one ScaI site for a single digest of ScaI found on column 3, the Plasmid A is
the pRSETB vector.
Table 1
Band
Lane
Migration Distance (cm)
Kb Pairs
Log (Kb
Pairs)
3
4
5
6
7
8
8
8
8
8
8
8
3.5
3.7
4.1
4.4
5.1
6.3
4
3
2
1.5
1
0.5
0.6
0.5
0.3
0.2
0.0
-0.3
Table 1 represents the values and measurements of the 1 kb ladder size marker, which exhibits:
the migration distance (in cm), the size of the fragment (in kb pairs), and the log of the kb pairs.
The data from bands 3 through 8 were used and matched up with their respective migration
distance and fragment size.
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Figure 2
Log (Kb Pairs) vs. Migration Distance of DNA
Ladder
0.8
Log (Kb Pairs)
0.6
y = -0.3115x + 1.616
R² = 0.97211
0.4
0.2
Log (Kb Pairs)
Linear (Log (Kb Pairs))
0.0
0
1
2
3
4
5
6
7
-0.2
-0.4
Migration Distance (cm)
The data from Table 1 was plotted into a graph (as seen above, on Figure 2) and was used to
derive the linear fit regression line equation to mathematically determine the sizes of the DNA
fragments of Plasmid A. The derived equation from Figure 2 is: log (kb)=-0.3135x + 1.616,
where x is denoted as the migration distance. To obtain the fragment size, the equation:
10log (kb) = kb, must be used. As seen above, the R2 value is 0.97211 for the linear regression line.
This R2 value expresses how closely matched the data points are in relation to the linear
regression line’s estimate of the equation.
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Table 2
Plasmid Fragment
Lane
Kb Pairs
1
1
2
3
4
Migration Distance
(cm)
3.7
5.6
3.5
3.5
3.7
2.9
0.7
3.4
3.4
2.9
Log (Kb
Pairs)
0.5
-0.1
0.5
0.5
0.5
BamH1 (Band 1)
BamH1 (Band 2)
Pst1
Sca1
BamH1 & Pst1 (Band
1)
BamH1 & Pst1 (Band
2)
BamH1 & Sca1
(Band 1)
BamH1 & Sca1
(Band 2)
BamH1 & Sca1
(Band 3)
BamH1 & Sca1
(Band 4)
Pst1 & Sca1 (Band 1)
Pst1 & Sca1 (Band 2)
Pst1 & Sca1 (Band 3)
Uncut
4
5.7
0.7
-0.2
5
3.6
3.1
0.5
5
4.2
2.0
0.3
5
4.9
1.2
0.1
5
5.7
0.7
-0.2
6
6
6
7
3.5
3.8
4.9
3.5
3.4
2.7
1.2
3.4
0.5
0.4
0.1
0.5
kb sum
3.6
3.4
3.4
3.6
7
7.3
3.5
The represented data in Table 2 exhibits the migration distances, fragment size, and the log (kb)
of the uncut Plasmid A, and the single and double digests that took place with the restriction
endonucleases. The log (kb) from both the single and double digestions were calculated using the
linear regression line formula derived from Figure 2, log (kb) = -0.3135x + 1.616, which in turn
gave us the fragment size (kb pairs). Sample calculation: log (kb) = -0.3135(PstI migration
distance) + 1.616 = (-0.3135)(3.5) + 1.616 = 0.5, then kb = 100.5 = 3.4 kb pairs.
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Figure 3
BamHI and ScaI
BamH1
pRSETB
3.9 kb pairs
ScaI
The above figure is the restriction map of Plasmid A. Through trial and error and deductive
reasoning, the Plasmid’s restriction sites were mapped.
Discussion and Conclusion
Deduced from Figure 1, the DNA from Plasmid A was the pRSETB vector. The most
prominent reason as to why it’s the pRSETB vector is because in lane 3, only one DNA fragment
showed up on the single digest with the ScaI endonuclease. It is impossible for it to be pQE30
because if it were, two DNA fragments would be seen in lane 3 caused by ScaI. In Figure 1, as
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seen in lane 1, two DNA fragments are seen meaning BamHI cut twice. Both PstI (lane 2) and
ScaI (lane 3) exhibited one DNA fragment of uniform fragment size according to Table 2 as 3.4
kb pairs. This meant that this was analogous to the parent DNA and was only cut once. A
discrepancy is observed in the single digests of both PstI and ScaI, so the double digests were
implemented to further characterize the specific restriction sites of these enzymes, which are
seen in Figure 1 and Table 2.
Double digestion of both BamHI and PstI yielded two fragments of 2.9 kb pairs and 0.7
kb pairs, respectively (seen in Figure 1). Observing, one of the BamHI single digest sites in
Table 2, there was also a 0.7 kb pair fragment which meant that both BamHI and PstI share a
common restriction site on the plasmid. In lane 4 (Figure 1), the double digest of BamHI and
ScaI produced four fragments (the largest being the parent DNA) with sizes of 3.1, 2.0, 1.2, and
0.7 kb pairs, respectively (seen in Table 2). Comparing the smallest fragment, 0.7 from this
double digest to the 0.7 from single digest of BamHI, this indicates that the two fragments are
analogous (seen in Figure 1 and Table 2). This meant that the other two fragments produced
(excluding the parent DNA band) were produced on the larger, 2.9 kb pair fragment. Lastly, the
double digest with PstI and ScaI yielded three fragments (the largest one being the parent DNA
fragment) with sizes of 3.4, 2.7, and 1.2 respectively (seen in Figure 1 and Table 2). By
observing Table 2, the 1.2 kb pair fragment from this double digest was analogous to the 1.2 kb
pair fragment produced by the double digest of BamHI and ScaI. It was deduced that the ScaI
restriction site was 1.2 kb pairs away from the PstI site (seen in Figure 1 and Table 2).
Analyzing the data from Figure 1 and Table 2 manifested Figure 3, which is the map of
the restriction sites for the BamHI, PstI, and ScaI endonucleases. Early on, the linear regression
line, which was derived from data pertaining to Table1’s migration distances of the lane 8 DNA
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ladder, gave rise to the fragment sizes seen on Table 2. The reason why bands 3 through 8 were
used in Table 1, was because band 3 was indicated as the parent DNA due to the PstI and ScaI
bands which only made one cut, yielding the largest length of possible DNA from the Plasmid.
Based off of the linear regression equation from Figure 2, the parent DNA fragment calculated
from the single band produced by the single digestion of PstI and ScaI yielded a DNA fragment
length 3.4 kb pairs. This value is also analogous to lane 7’s uncut DNA which yielded a fragment
length of 3.4 kb pairs based on Table 2.
Ultimately, this experiment was deemed a success. Despite the success, there was
definitely a discrepancy in the size of the plasmid. According to Figure 1, the plasmid size is 3.4
kb pairs whereas according to the restriction map of Figure 3, the plasmid size is 3.9 kb pairs.
This discrepancy could have arisen from many variables. These variables may include:
accidental error; from the possibility of adding a slightly incorrect amount of plasmid and
endonucleases, not keeping the samples in the incubator long enough, a discrepant concentration
of agarose which wasn’t optimal for the samples, or possibly not letting the samples
electrophorese long enough. For future reference, these possible errors will be taken into account
and monitored to produce an accurate, non-discrepant experiment.
References
1. Cox, Michael L., and David L. Nelson. Lehninger Principles of Biochemistry 6th Ed. New
York: W.H. Freeman, 2013.
2. Brooks, Joan E. "Properties and Uses of Restriction Endonucleases." Restriction
Endonucleases 11:113-129.
3. Adams, Deborah A, and Richard C. Ogden. "Electrophoresis in Agarose and Acrylamide
Gels." Gel Electrophoresis, no. 8, 61-87.
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