Download The Polymerase Chain Reaction

Polymerase Chain Reaction Preparation & Amplification
Procedure:
1.
A 40ml centrifuge tube was gathered and labeled with a permanent marker.
2.
Next a small plastic cup was gathered and filled with approximately 10ml of 0.9%
sodium chloride.
3.
The saline solution was placed in the mouth and swished for 30 seconds to clean the
mouth of food particles and bacteria, then it was expelled into the sink. The bacteria
could cause wrong results and the food particles could clog the pipette tip.
4.
After that 10ml of 0.9% saline solution was placed in the mouth and swished for 30
seconds to gather cheek cells. Then the saline mouthwash was expelled in the plastic
cup.
5.
Then the saline mouthwash was poured very carefully a 40ml centrifuge tube. All
appropriate precautions were used as to not cause any cross contamination from the
mouthwash.
6.
Next the tube was centrifuged at 12,000rpm’s for 2 minutes. This produced a cell pellet
at the bottom of the tube.
7.
The saline solution was then poured out of the tube making sure not to disturb the cell
pellet. Then the tube was filled with 1ml of 0.9% saline solution to resuspend the cells
and liquefy the pellet to make a cell suspension.
8.
After that a 1.5ml centrifuge tube was gathered. Then using a 1000l micropippetor,
200l of InstaGene matrix along with 30l of cell suspension, were added to the 1.5ml
2
centrifuge tube. The InstaGene matrix provided the PCR ready DNA, and the purified
DNA preparations.

Prior to this step in the experiment the InstaGene matrix was mixed at a moderate
speed to keep it in a liquid form.
9.
Next the 1.5ml centrifuge tube was incubated at a temperature of 56OC for 20 minutes.
This lyzed the cells, causing them to burst and spill their contents, along with the DNA.
10.
After incubation the tube was then vortexed for 10 seconds at a high speed to remove the
cell debris from the solution.

Next the tube was placed in a water bath with a temperature of 100OC for 8 minutes.
This denatured the enzymes and stopped the lyzing of the cells.
11.
Then the tube was taken out of the water bath and vortexed for 10 seconds at a high
speed.

12.
Then spun for 2 – 3 minutes at 12,000rpm’s.
All of the subsequent ingredients were pre-chilled, except the resulting supernatant. And
all were added to a 1.5ml centrifuge tube in no particular order.

45l
PCR MasterMix
1l
Primer solution
18l
Template DNA solution
The PCR MasterMix was at a 1.1X concentration, and contained deoxyribonucleotide
triphosphates, recombinant Taq DNA polymerase and Mg++. The Taq DNA
polymerase was there to synthesize new strands of DNA and it was an enzyme that
was able to withstand the high heat. The Taq was isolated from the bacteria Thermus
3
aquaticus. The purpose of the PCR MasterMix was to provide the reagents needed
for the amplification of the template DNA.

The primer solution (17-mers) was used because it allowed specific binding. It also
allowed for only a possibility of one binding site to the human DNA.
13.
Next the tube was pulsed for 10 seconds in a centrifuge to thoroughly mix it.
14.
Using a 20l micropippetor, 10l of mineral oil was added to the tube. The mineral oil
acted as an evaporation barrier when the tube was placed into the thermocycler with a
high temperature.
15.
After that the tube was closed and loaded into the thermocylcer with a temperature of
80OC.
16.
Then the cycling program file was started. Thirty cycles were done. Each cycle was as
follows:
94OC for 1 minute
55OC for 2 minutes
72OC for 2 minutes

The 94OC was when the DNA was denatured into single strands.

The 55OC was when the primers anneal on either side of the target region to their
complementary sequences.

The 72OC was when the polymerase binds and makes a complementary DNA strand
to the single stranded DNA.
17.
Next Electrophoresis began.

A 2.0% Agarose gel was poured into a gel-casting tray at a depth of 5mm.
4
18.
While the gel was solidifying a clean 1.5ml centrifuge tube was gathered and labeled
with a permanent marker with the group’s name.
19.
Making sure not to take any of the mineral oil up, a micropippetor was used to pipette
10l of the amplification product from the PCR reaction.
20.
Next 5l of loading dye was added to the tube. Then the tube was centrifuged.
21.
After the gel had solidified the gel-casting tray was placed into the electrophoresis box
with TBE buffer, and the comb was removed.

10l of the PCR reaction was added to the wells as follows:
B+H

Unamplified
control
Student Samples
1
2
3
4
5
6
The B+H stands for /BamHI+HindIII. This was here to provide a size marker.
Since it produced 493bp and 125bp bands, the student samples could be compared
and checked that they were in the expected size range. That expected size range
being 400bp and 100bp.

The unamplified control was to provide the assurance that the PCR products seen in
the student samples, the amplified ones, were truly PCR products.
22.
After that the gel was electrophoresed at 100 volts for 30 minutes.
23.
Then the power was turned off and the gel-casting tray was removed from the
electrophoresis box and stained with ethidium bromide for 20 – 30 minutes, then rinsed.
24.
And lastly a picture was taken with an ultraviolet transilluminator, and viewed.
5
Results:
For an unexplained reason the gel for the group did not turn out correctly, so the ideal gel
was used for the results and discussion sections of this paper. For the 3rd, 4th, and 6th wells,
under student samples, only one band was visible. For students 1,2, and 5, two bands were
visible. The well that was labeled B+H, produced six bands that were visible. The unamplified
control produced no visible bands.
For students 1, 2, and 5 there were two bands visible, that meant that these people were
heterozygous (+/-) for the TPA-25 Alu insertion. The student that showed a homozygous trait
(-/-) for the absence of TPA-25 Alu insertion, was the 3rd student. Two students showed
homozygous for TPA-25 Alu insertion (+/+), they were students 4 and 6. Therefore, there were
two students who were homozygous (+/+) for TPA-25 Alu insertion. There were a total of 3
students who were heterozygous (+/-) for TPA-25 Alu insertion. Then there was only 1 student
who was homozygous (-/-) for the absence of the TPA-25 Alu insertion.
For the presence of TPA-25 insertion the allelic frequency was found to be 0.58. The
allelic frequency for the absence of TPA-25 insertion was found to be 0.42. Both these numbers
were used to equate the Hardy Weinberg equation (p2 + 2pq + q2 =1). The allelic frequencies (p
and q) were represented by p=0.58 and q=0.42. The p2 and q2 were found by squaring the p and
q, they were found to be p2=0.34 and q2=0.17. These numbers were then plugged into the Hardy
Weinberg equation as follows: 0.34 + 2(0.58)(0.42) + 0.17, with the answer being 0.93.
Discussion:
The region of DNA that was used for this experiment was from chromosome 8. This part
of the DNA had a short sequence known as Alu. This short sequence was in the tissue
6
plasminogen activator (TPA) gene. There were approximately 500 to 2,000 Alu sequences in the
human genome. The particular Alu used in this experiment was the TPA-25, which was found
inside of an intron of the TPA. This gene is not found in all humans. This was where the
polymerase chain reaction (PCR) came into play. Using PCR, people who had this particular
TPA-25 insertion were screened. If the TPA-25 insertion was present than the bands should be a
size of 400bp. If the TPA-25 insertion was absent than it should be only 100bp long. This was
why the B+H was used, because the B+H produces 493bp and 125bp.
If there was only 1 band visible, as with students 3, 4, and 6, this meant that they were
homozygous for the TPA-25 Alu insertion. For student 3, the band was 100bp long, meaning
that this individual was homozygous for the absence of the TPA-25 Alu insertion. For students 4
and 6, the bands had lengths of 400bp, meaning that these individuals were homozygous for the
presence of the TPA-25 Alu insertion. Two bands were visible for students 1, 2, and 5. All three
students were heterozygous for the TPA-25 Alu insertion. If no bands appeared this meant that
there was an error during the preparation of the PCR samples, but this did not occur during this
experiment. Also three bands would mean that the primers did not bind specifically to the
chromosomal loci, this did not occur during this experiment.
The allelic frequencies were ratios that compared the number of copies of a certain allele
to the entire number of alleles present in the experiment, which was 12. The allelic frequency
for the presence of the TPA-25 Alu insertion was calculated as follows:
[2 X 2(homozygous) +3(heterozygous)] / 12 = 0.58. The absence of the TPA-25 Alu insertion was
calculated as follows: [2 X 1(homozygous) + 3(heterozygous)] / 12 =0.42. Next the distribution of
genotypes for the class was calculated using the Hardy Weinberg equation. The allelic
frequencies that were calculated above were used for this equation as p and q. When the
7
numbers were plugged in the answer was 0.93. The Hardy Weinberg equation usually equals 1,
meaning that a population was in equilibrium because the allelic frequency doesn’t change from
generation to generation. But the human species is not genetically stable enough for the Hardy
Weinberg equation to equal one. Also the distribution of genotypes that were calculated was not
right was because not a large enough population was used. For this experiment only 6 students
were used. If a proper calculation was needed, than a larger population would be needed in order
to get valid data.