PCR and agarose gel electrophoresis

Introduction

PCR (polymerase chain reaction) is an in vitro technique enabling researchers to

produce millions of copies of a specific DNA sequence in approximately 2 hours
inside a test tube. This automated process bypasses the need to use bacteria for
amplifying DNA.

To perform a PCR, we will need a reaction mixture containing the target DNA
sequence to be amplified, 2 primers (forward and reverse), heat-stable Taq DNA
polymerase and deoxynucleotide triphosphates.

The first step of the cycle is denaturation, brought about by heating the target DNA
to about 95°C. This process separates the double-stranded DNA into 2 single
strands. After strand separation, cooling of the DNA in the presence of a large
excess of forward and reverse primers allows these primers to anneal and hybridise
to complementary sequences in the 2 DNA strands. The optimal temperature for this
to occur varies between 40°C to 60°C.

The mixture is then incubated with Taq DNA polymerase at 72°C which enables
nucleotides to be added to the 3' end of the annealed primers and extend in the 5'
to 3' direction.

The temperature is raised to denature the DNA into 2 single strands again and then
lowered sufficiently to allow more primers to anneal. Taq DNA polymerase now
synthesises another set of new complementary strands.

As the procedure is repeated over and over again, the newly synthesised fragments
serve as templates for the next cycle and within a short period of time, many copies
of the original DNA can be produced. This results in an exponential accumulation of
the specific target fragment, approximately 2n where n is the number of cycles of
amplification performed.

Gel electrophoresis is a widely used technique for the analysis of nucleic acids and
proteins. Agarose gel electrophoresis is routinely used for the preparation and
analysis of DNA. Gel electrophoresis is a procedure that separates molecules on the
basis of their rate of movement through a gel under the influence of an electrical
field.

DNA is negatively charged and when placed in an electrical field, DNA will migrate
towards the positive pole (anode). An agarose gel is used to slow the movement of
DNA and separate by size. Within an agarose gel, linear DNA migrates inversely
proportional to the log10 of their molecular weight.

Aim

The aim of this experiment is to identify the absence or presence of the human
“transgene” in 2 samples of mouse genomic DNA using the PCR method.
Materials and Method

Target DNA – I will be using 2 different samples of mouse genomic DNA

alongside a dH2O control sample which will be used as a negative control.

Buffer solution – This is used to provide a suitable chemical environment for

optimum activity and stability of the DNA polymerase.

Deoxynucleotide triphosphates – These are used in excess for which the DNA
polymerase uses to synthesise a new complementary DNA strand.

MgCl2 - MgCl2 is a cofactor of Taq DNA polymerase, the concentration of MgCl2

influences the productivity and fidelity of polymerases. At optimal concentration,
that must be determined for each primer/template condition. It is typically
between 1.0 and 3.0 mM.

Two oligodeoxynucleotide primers - The two primers, each typically about

15-30 nucleotides in length, are usually designed so they are 200-2000 bp
apart, one hybridizing to one strand of dsDNA, the other hybridizing to the other
strand such that both primers are oriented with their 3' ends pointing towards
each other.

Taq DNA polymerase – This thermophilic DNA polymerase is thermally stable

and is able to work at 100°C at which DNA is denatured into linear strands. Taq
DNA polymerase has an optimum temperature of 72°C.

1. The reagents are mixed together and centrifuged - dH20, 10x NH4 PCR
buffer, 50 mM MgCl2, 50uM Forward primer, 50uM Reverse primer, 10mM
dNTPs, Taq polymerase (5u/ul).

2. 24ul of the mixture is aliquoted into 3 eppendorf tubes.

3. In the first tube, 1ul of genomic DNA1 was added. In the second tube 1ul
of genomic DNA2 was added and in the third tube, 1ul sterile water was
added.

4. The tubes are then left in ice to be placed into an MJ Research PCR
thermal cycler and stored at -20oC for the next part of the experiment.

5. During the next part of the experiment – gel electrophoresis, 1% (w/v)

agarose gel was prepared by mixing 1g of agarose with 100mls of 1X TBE
buffer in a 250ml flask.

6. This mixture is then heated carefully in a microwave up to its boiling point

with occasional gentle swirling.
7. The agarose gel is left to cool before 2.5ul of 10mg/ml ethidium bromide
solution per 100ml gel was added and mixed gently. This allows the DNA
to be visualised under UV light.

8. The ends of the gel former were sealed with tape and the agarose gel is
poured into it and left to set.

9. Once the gel has set, tape is removed and placed into an electrophoresis
tank and 1X TBE buffer is poured into the tank until it is about 1m above
the gel.

10. 5ul of the loading dye is added to each of the 3 eppendorf tubes. This
aids the loading into the wells by increasing the sample density and allows
us to see how far the samples migrate.

11. 5ul of Hyperladder I DNA size marker is loaded into a well and 20ul of
each of the PCR samples are loaded into the wells beside it.

12. The voltage on the tank is then turned on so that electrophoresis can
occur. The electrophoresis is stopped before the dye runs of the end of the
gel. Gels are then viewed on a UV transilluminator and a photographic
image is captured to be analysed.
 Results

A photographic image obtained from our group (Group 19)

Distance
migrated from

200bp

Hyperladder I
DNA molecular dH2O (Negative
weight marker Control)

Sample 1 Sample 2
(Figure 1)

(Figure 2)
Distance migrated by
Sample 2 – 6.1cm
Discussion
From looking at the photographic image (Figure 1), we can see clearly that
Sample 2 of mouse genomic DNA has produced visible PCR products of the
correct size which is just slightly above the DNA molecular weight marker of
200bp.

In this experiment, we have used human-specific primers (forward and

backward) which amplifies a segment of human DNA at 227bp. Therefore we
have identified that only Sample 2 shows the presence of a human “transgene”
sequence, whereas in DNA Sample 1 there are no distinct amplications which
therefore shows us the absence of such “transgene”. The negative control shows
no result as expected as there was no DNA sample present; this also shows that
there was no potential contamination which could have affected the PCR results
obtained.

Looking at the graph (Figure 2), a relationship between the DNA molecular
weight and the distance it travels can be clearly seen.
We can also see a negative correlation as the distance migrated increases, the
molecular weight of the DNA decreases. The graph almost shows a straight line
which also assumes that the relationship is also inversely proportional.

I have measured the distance in which the PCR products of Sample 2 have
migrated which is 6.1cm. This is indicated on the graph and the approximate
molecular weight of the DNA can be determined. From the graph, it shows that
the molecular weight in log10 is 2.4bp. To convert this, we take the inverse log of
2.4 and we get 251bp for DNA Sample 2. This is a rough estimate which is in
close proximity to our value 227bp that we are looking out for. Therefore this
has proved we have shown the presence of the human “transgene” of the
correct size.

The distance DNA molecules can travel through agarose gel depends on the size
of DNA molecules in electrophoresis. The agarose gel acts as a sieve for DNA
molecules so that larger molecules have difficulty moving through the gel matrix
compared to smaller molecules which can move more freely. Therefore, smaller
fragments of the DNA are able to migrate further through the gel towards the
positive end as DNA is negatively charged. This process allows the separation of
large and small DNA fragments which can then be analysed and identified.

We have used a molecular weight marker alongside our DNA samples which is a
mixture of DNAs with known molecular weights; we can therefore use this
marker to estimate the size of our DNA fragments.

In conclusion, I have successfully shown that the PCR had worked in which the
sample of mouse genomic DNA containing a human “transgene” had been
identified and that the electrophoresis results have given me a set of good
reliable results.