Introduction: and amplifying specific regions of DNA.

 

Introduction:

Polymerase chain reaction (PCR) is a
popular laboratory technique of producing copies and amplifying specific regions
of DNA. PCR was recognised in 1993 for being an important research tool and was
awarded the Nobel Prize in chemistry to Kary Mullis. 1 The technique applies basic knowledge of DNA
replication such as using DNA polymerase to synthesise new strands. For PCR the
DNA polymerase typically used is Taq polymerase, an isolated bacterium with
heat stability. The Taq polymerase extends the existing strand of DNA with the
addition of deoxy nucleotides (dNTPs). The complementary dNTPs are added onto
the strand that is being copied. PCR primers are used to define the starting
point of replication. Primers are a short sequence of nucleotides complementary
to the specific sequence of the DNA strand which is to be copied. Two primers
are used as they must bind to opposite strands of the template DNA (shown in
figure 1). 2

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Figure 1 Polymerase chain reaction (2017) 2 this illustration shows 2 different primers
binding to opposite strands on the template DNA. Notice they are binding to
the template strand via complementary base pairing.

 

 

 

As for the PCR cycle it consists of
three basic steps for the DNA to be synthesised. Denaturation which happens at
around 96oC where the DNA strand separates out into 2 single
strands. Annealing occurs between 55-65oC where the DNA is cooled
and primers are able to bind to the template strand. Finally there is extension
at 72oC where the reaction temperature increases allowing the Taq
polymerase extend the primers, making new DNA strands. These 3 steps are
considered 1 cycle and each cycle can generate twice as many copies (figure 2). 2

 

 

 

 

 

 

Figure 2 Polymerase chain reaction (2017) 2 depicts the exponential growth of the template
strand synthesising twice as many new DNA strands per cycle.

 

 

 

In this practical we used a primer
pair that can amplify inserts in standard cloning vectors such as plasmid DNA. The
primers used in the practical have the following sequence of:

M13forward
(5′-CCAGGGTTTTCCCAGTCACG-3′)

M13Reverse (5′-TCACACAGGAAACAGCTATG-3′)

Using three unknown plasmids the
primers will bind to opposite strands of the plasmid DNA. These primers will magnify
the specific insert without the rest of the DNA. The three unknown plasmid templates
being used are all based on the same cloning vector, pBluescript which is roughly
3 kb long. The plasmids differ in the length of their inserts: cSox1 contains a
~0.3 kb insert, cWnt1 contains a ~0.6 kb insert and cMyoD contains a ~1 kb insert. 1

In order to visualise the results
from the PCR we use gel electrophoresis to separate the DNA into fragments
which are pulled by an electric current through an agarose gel. Along with the
three unknown plasmids there is also a negative control which ensures us if there
is any contamination of the reagents. To determine the length of the plasmid
inserts there is a DNA molecular weight marker (1 kb ladder) which we use to determine
the size of visible DNA fragments.

The application of PCR has shown
the technique to be incredibly useful and efficient at identifying, isolating,
mapping, and sequencing the human genome.
3 Other fields of interest include amplifying genes associated with
genetic disorders from the DNA of patients. This can be performed on foetal DNA
to determine whether or not they carry a genetic disorder.

Specific experimental skill aims
include using the PCR method to produce copies and amplify inserts of three unknown
plasmids. To be able to identify and analyse the results with the use of gel electrophoresis.
This technique will allow us to determine which unknown PCR product correlates
to the given plasmid and its insert.

Method: 1

PCR Reaction

In 3 PCR tubes each labelled either
1, 2 or 3 the following solutions were pipetted: 9.5 µl H2O, 12.5 µl
2x BioMix Red (Taq polymerase, dNTPs, buffer), 1 µl M13F primer (50 µM), 1 µl
M13R primer (50 µM) and 1 µl of the appropriate plasmid DNA making sure it
matched the labelled PCR tubes. Along with the three PCR tubes there was a fourth
PCR tube labelled N for negative control. The same volume of each solution was
pipetted into the tube, however the 1 µl of plasmid DNA was replaced with an
additional 1 µl of H2O. All four tubes were placed in the PCR
machine and the following programme was ran: the initial denaturation for 1
minute at 95oC, 25 cycles of denaturation for 15 seconds at 95°C, primer
annealing for 15 seconds at 57°C, polymerase extension for 1 minute at 72°C and
there was the final primer extension step of 5 minutes at 72°C. Approximately
this thermal cycle took 45 minutes to complete.

Gel electrophoresis procedure

With a gel premade it was placed in
the gel tray and the TBE buffer was added to cover the gel. 12 µl of the DNA
molecular weight marker (1 kb ladder) was pipetted into the first well. Following
the first well, 8 µl aliquots from each PCR tube were pipetted in the order of
N, 1, 2, 3. The loaded electrophoresis tank was connected to the power supply
and the gel was ran at 120V for approximately 40 minutes or until the red dye
was about 2cm from the bottom. The gel was then removed from the tank and
visualised using a UV lamp.

 

 

 

 

 

 

 

 

Results:

 

 1         
2        3        4          5         6

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3 Gel electrophoresis UV
image of the inserts amplified from the three unknown plasmids. 

Gel loading order:

Lane 1 – molecular weight marker (1
kb ladder)

Lane 2 – negative control

Lane 3 – PCR product 1

Lane 4 – PCR product 2

Lane 5 – PCR product 3

Lane 6 – 100 base pair ladder

The resulting plasmids were separated
by gel electrophoresis with each insert amplified as shown in Figure 3. By
using the 1 kb ladder I figured out the lengths of each PCR product. PCR
product 1 was ~0.6 kb, PCR product 3 was ~1 kb and as for PCR product 2 there
were no visual bands.

Discussion and Conclusion:

We were given three different
cloning vectors each with a varied size insert. The aim of the experiment was
to use the PCR method to produce copies and amplify inserts of the three
unknown plasmids. Using gel electrophoresis, we were able to identify which PCR
product correlates to the given plasmid and its insert. The plasmid cWnt1
contained a ~0.6 kb insert which meant out of the three unknown plasmids it was
plasmid 1 as the band in lane 3 is just above the 0.5 kb marker on the ladder.
cMyoD contained a ~1kb insert deducing that it was in fact plasmid 3. As for cSox1
which contained a ~0.3 kb insert must have been plasmid 2, but for possible
reason of error there were no visible bands in lane 4.

The aims of the experiment were to
identify the template plasmids on the base of the size of the insert even
though there was some error with plasmid 2. Given correct PCR conditions we
should have expected 1 band in lanes 3 and 5, however analysing figure 3 there
shows a possible extra 2-3 faint bands present. There are multiple reasons
which could indicate the reason for extra bands in the lanes. An example could
be due to a primer dimer which is a potential by-product where primers
hybridise to one another due to complementary bases in the primers. 4 I don’t believe there
was any contamination as we see in lane 2 the negative control is clean.

As I mentioned previously with
plasmid 2 not being amplified, this unexpected result may have been caused by not
pipetting the plasmid from the eppendorf tubes correctly. In future experiments
to prevent this from happening I could centrifuge the samples prior to placing
them into the thermal cycler machine. This would ensure thorough mixing of the
sample, so when loading the sample into the wells there is DNA present.

Future directions with this experiment has seen it
to be a reliable and simple way to create recombinant DNA. 5 Recombinant DNA is artificially formed DNA that is
combined from two nonhomologous DNA.
6 The outcome of this experiment meant the aims were met and the
experiment was largely a success, with the unexpected results for cSox1 plasmid
becoming very informative for how we can improve the experiment with additional
techniques.