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128
2010
Abstract
Recombinant DNA technology was used to clone specific DNA fragments. Before the technology was used, a basic
understanding of the parts and details of the procedure were investigated. It was found that the larger the genome of an
organism, the greater its overall complexity relative to other living organisms. This was discovered through the comparison
of various genome sizes coming from known specimen. Gel electrophoresis technology was utilized to compare the relative
sizes of the DNA. The gels were run from left to right with increasing level of complexity. The samples were, from left to
right, plasmid DNA, lambda bacteriophage DNA, calf thymus DNA, and calf kidney DNA. As the complexity of the DNA
increased, the number of bands in the lane increased to the point that the Eukaryotic DNA found in the calf thymus and
kidney were smears. The gel displayed identical smearing and satellites for the calf kidney and thymus DNA, proving that the
DNA is the same even though they are from different part of the organism. Through DNA fingerprinting, DNA fragments
were determined that matched a specific pattern after being exposed to a restriction enzyme (HindIII). The DNA fragment
containing the 2.0 fragment was determined by comparing the electrophoresed cut DNA to known standards and also
observing that two highly conserved sequences were seen on the gel after electrophoresis. Through the use of ligation and
transformation, DNA cloning was accomplished. Tests were run that involved electrophoresis and digestion by HindIII in
order to fingerprint the DNA. The clone was identified as the desired sample through the use of transformation to identify
white colonies (those with the insert) and blue colonies (those lacking the insert). The DNA molecule desired was then
isolated through the use of a special resin that binds to DNA at high salt and not at low salt. The DNA is purified and the
cloned sample is obtained. To verify that the cloned sample contains the correct ligating insert, digestion of the DNA was
performed with HindIII and electrophoresed. The samples were found to contain the 2.0 kbp lambda fragment verifying that
the sample was indeed cloned by recombinant DNA technology.
Key words: DNA Technology, Cloning, Genetics, Biotechnology.
Introduction
Over the course of this lab experiment, recombinant
DNA technology was used to clone specific DNA
fragments. There were several steps to this procedure that
utilized various forms of technology and information.
The steps performed involved analysis of the anatomy
and evolution of the genome, genetic fingerprinting of
an unknown sample, the recombinant DNA technology
which can be split into ligation, fingerprinting, miniprep,
and digestion (Leisner and Shemshedini, 2009).
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Results
The results that will soon be presented are used
to either refute or support the hypothesis that using
recombinant DNA technology allows for the cloning of
specific DNA fragments. Several tests were performed in
order to test this hypothesis. The anatomy and evolution
of the genome was under study. Genetic fingerprinting
2010
Figure 1. Photograph of UV Transilluminated Electrophoresed Gel of Plasmid DNA, Lambda bacteriophage DNA, Calf Thymus
DNA, and Calf Kidney DNA
T K
23.1 kb
9.4 kb
6.6 kb
1.44 kb
4.4 kb
2.77 kb
2.3 kb
1.44 kb
P = Plasmid DNA
2.0 kb
0.56 kb
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Table 1. Distance Migrated and Size of DNA Molecules for Standards, Calf DNA, and Plasmid DNA
Distance Migrated
by Standards (cm)
Sizes of DNA
molecules in
Standards
3.7
1.36
23.1
4.5
0.97
9.4
5.0
0.819
6.6
Plasmid DNA
7.2
0.443
2.77
5.8
0.643
4.4
8.6
0.158
1.44
7.3
0.361
2.3
Calf thymus
DNA (satellite)
7.5
0.301
2.0
8.6
0.158
1.44
11.1
-0.252
0.56
Sample
Distance
Migrated
(cm)
DNA Fragment
Length Log
Kilobase Pairs
Sizes of DNA
Molecules
(kb)
Graph 1. Lambda Bacteriophage DNA Log Base 10 of Molecular Weight versus Distance Migrated
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2010
DNA Fragment
Length Log Kilobase
Pairs
Molecular
Weight
(kbp)
Cut HSHV 1
4.5
0.301
Cut HSHV 2
4.3
0.431
2.7
Cut CHAV
3.6
0.672
4.7
Uncut HSHV 1
3.1
0.877
7.5
Uncut HSHV 2
3.9
0.561
3.6
Uncut CHAV 1
3.3
0.798
6.3
Uncut CHAV 2
4.2
0.443
2.8
Uncut KDV 1
3.1
0.877
7.5
Uncut KDV 2
3.9
0.561
3.6
Cut KDV 1
4.5
0.301
Cut KDV 2
4.3
0.431
2.7
Molecular Weights
of Standards (kbp)
Distance Migrated
(cm)
23.1
3.7
1.36
9.4
4.5
0.97
6.6
5.0
0.819
4.4
5.8
0.643
HU = HSHV Uncut
2.3
7.3
0.361
7.5
0.301
0.56
11.1
-0.252
HU HC KU
KC CU
CC
Sample
6.3 kbp
7.5 kbp
2.7 kbp
2.7 kbp
7.5 kbp
4.7 kbp
3.6 kbp
3.6 kbp
2.8 kbp
2.0 kbp
2.0 kbp
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Graph 2. The Standards from Graph 1 used to Compare Against to Discover the Molecular Weight of KDV Virus Fragments
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T2
T3
T4
12
N/A
N/A
N/A
Number of
colonies
Color of
colonies
T2 (LB)
2010
Smeared
Blue and
White Satellite
Not
Distinguishable
1
Blue
White
White
White
White
White
S = Standard DNA
BU = Blue Colony Uncut
BC= Blue Colony Cut by HindIII
W1 = White Colony Cut by HindIII
W2 = Different White Colony Cut by HindIII
W3 = Different White Colony Cut by HindIII
W4 = Different White Colony Cut by HindIII
W5 = Different White Colony Cut by HindIII
DNA Fragment
Length Log
Kilobase Pairs
Sizes of DNA
molecules (kb)
3.1
0.699
3.3
0.602
3.7
0.477
3.9
0.398
2.5
4.3
0.301
4.8
0.176
1.5
5.8
6.4
-0.125
0.75
7.2
-0.301
0.5
8.2
-0.602
0.25
Sample
Standards
2010
Distance
Migrated (cm)
DNA Fragment
Length Log
Kilobase Pairs
Sizes of DNA
molecules (kb)
Sample
N/A
N/A
N/A
Blue Uncut
4.1
0.392
2.7
Blue Cut
0.416
2.7
White 1
4.4
0.321
2.0
White 1
0.416
2.7
White 2
4.4
0.321
2.0
White 2
0.416
2.7
White 3
4.4
0.321
2.0
White 3
0.321
2.7
White 4
4.4
0.321
2.0
White 4
0.416
2.7
White 5
4.4
0.321
2.0
White 5
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Table 6. The Characteristics and Plasmid Names of the Different E. coli Strains Created
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Calculations
For each lab standard graph, the log base 10 was taken
of the molecular weight and plotted against the distance
migrated from the point of insertion.
Example (From Table 1)
log (molecular weight) = log base value
log (23.1) = 1.364
To determine the molecular weight of the unknown
samples being tested in each case, the distance migrated
values were plugged into the trend line equation and
that value was then used as a power of ten to obtain the
molecular weight. The example is taken from the plasmid
DNA in Table 1.
Example (From Table 1)
x = distance migrated (cm) y = log base 10 value of
molecular weight
y = -0.204x + 1.912
= -.204(7.2) + 1.912
= 0.443
To find the molecular weight the log base 10 value is
taken as a power of ten.
Molecular weight = 10(log base 10 value)
= 100.443
= 2.77 kbp
Discussion
The hypothesis for the entire experiment that
was performed was that by using recombinant DNA
technology, one can clone specific DNA fragments. The
results were used to determine whether or not this claim
is supported.
In testing the anatomy and evolution of the genome,
the larger the genome size the more bands were found
when the DNA was electrophoresed. In the example found
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References
Kornberg, A., & Baker, T. (2005). DNA Replication.
University Science Books.
Leisner, S. & Shemshedini, L. (2009). Fundamentals
of Life Sciene I Lab Honors.
Reddi, O.S. (2000). Recombinant DNA Technology.
Allied Publishers.