Result Polymerase Chain Reaction (PCR)

Notice the solution within the microcentrifuge tube after performing PCR. Test Tube Sample Negative Control Change Noticed Clear Solution Clear Solution

The solution from the microcentrifuge tube is then taken for analysis by Agarose gel electrophoresis. Notice the bands that form on the agarose gel under the UV transaminator. Test Tube Sample Negative Control Change Noticed A single band appeared on the gel No band was formed

Conclusion and Discussion

PCR is an imitation of natural DNA synthesis. However, contrary to natural DNA synthesis, PCR is performed within test tubes, this allows one to select a desired segment of DNA and synthesize the amount wanted.

When performing PCR, it is important to know the nucleotide sequence desired. The nucleotide sequence will be taken to construct a primer, which are small oligonucleotides with a base sequence capable of binding to specific single stands of DNA. Therefore, it is necessary to use 2 types of primers or a pair for each single stand of desired DNA. It is adamant that the primers work in opposite directions, do not bind to each other, and bind to DNA at a position that is far enough from the other strand (In the experiment we used primers with base lengths 200 and 100, with the binding point of each primer being 180 bases apart).

Requirements for PCR

1.

DNA template : DNA one wishes to increase DNA polymerase : enzyme in DNA synthesis that catalyzes the reaction that binds the new nucleotide with the primer. In the experiment, Taq DNA polymerase was used. This DNA polymerase belongs to bacteria (thermus aQuaticus) and is capable of enduring high temperatures. Therefore, it will not denature in high temperatures, in the step of primer extention (70-75 C)

2.

3.

dNTPs : the 4 types of deoxyribonucleotide triphosphate are used as substrates

4.

DNA primers : -Forward primer (5 primer) : will have the same sequence and direction as the target DNA used to design the DNA template
-

Reverse primer (3 primer) : will have a

sequence in a different direction that is complementary with the target DNA used to design the DNA template
5.

Buffer & Cofactor : solvent used to control and maintain the conditions under which the reactions occur ie. pH and Mg the cofactor of DNA polymerase will also be present
2+

* In PCR ; Tm is the temperature at which the primer will bind to the DNA template in the process of annealing Tm can be found from the following equation: Tm = 2(T+A) + 4(G+C)

Tm will help determine the appropriate temperature for annealing.

Annealing temperature should have value Tm 5 C .

This is an appropriate temperature for the primers as primers bind better at lower temperatures.

The Tm of both primers should have similar values (If their values should greatly deviate from one another, we wouldnt be able to control the temperature in the process of annealing).

In this experiment, we mixed the required solutions using the method of Master mix (which is simultaneously mixing all required solutions and pipetting the necessary quantities of each in desired ratios).
. l

The resulting mixture gives a total volume of 35

size l

We now pipette the mixture to two 0.2

microcentrifuge tubes. Each tube will contain 17.5

. Following

that, we add DNA template to one of the microcentrifuge tubes. This tube will be known as the sample tube. The other tube will be the Negative Control. Distilled water is added to the Negative Control. After that, 0.5
l

of Taq DNA polymerase is added to

each test tube. Next, both tubes are inserted into the Thermal cycler and we proceed to set up the programme. We set the time

and temperature of each step as required: Denaturation (the step used to start the cycle), Amplification (the step used to repeat the cycle as needed. In the real experiment, we repeated the cycle 30 times. The cycle consists of all denaturation, annealing and extention.), and Extention (the step used to end the cycle). Once PCR is completed, take the solution for DNA analysis using Agarose gel electrophoresis. PCR consists of 3 steps:
1.

Denaturation : separate the double stand of DNA into single strands using heat at a temperature of about 92 C (In the

real experiment, 95 C was used). This will destroy the

Hydrogen bonds holding the two DNA strands together.

2.

Annealing : Lower the temperature to 50-55 C (In the real

experiment, 65 C was used). The added DNA primer will

anneal with the singe strand of DNA at its complementary base starting at the 3 end of each strand.
3.

Primer extention : this is the process of synthesizing a new DNA strand relying on the 2 single strands of DNA as a template and synthesizing away from the primer in the 5 to

3 direction. This process relies on DNA polymerase and all 4 added deoxyribonucleotides (dATP, dTTP, dCTP, dGTP). DNA polymerase will bind the free bases with the primer. Each complementary base will bind with the template at the appropriate temperature for the catalysis of enzyme DNA polymerase which is around 73 C (In the real experiment,

72 C was used).

Once the reaction is done, completed cycle, you will have twice the amount of DNA that you started off with. The final result is a DNA strand with one strand from the template and the other strand from the primer. Because of this, this form of DNA replication is called Semiconservative DNA Replication. Each cycle will yield twice the amount of DNA that you started off with. Therefore it increases at an Exponential rate, 2
n

(n =
n

number of cycles). In the experiment, we performed 20-40 cycles. We can find the total amount of DNA from the equation (2 -2n)x. However, in the real experiment we will simply use 2 .
n

In the real experiment, we completed a total of 30 cycles, using formula 2 we can find the total amount of DNA.
n 30 Total DNA (new and old) = 2 = 1,073,741,824 DNA

DNA from PCR cannot be seen by the naked eye. Therefore DNA analysis will be done by Agarose gel electrophoresis. From our experiment, using the UV transilluminator, we were able to see a singe band on the gel. The band only appeared on the sample from the Sample test tube. No band appeared on the sample from the Negative Control. There is no band because we did not add DNA template to the Negative control. Instead, the Negative control consisted of distilled pair we added had nothing to anneal to (in the process of

water. Since there was no DNA template, the first primer annealing). Therefore there was no increase in DNA. With appear in the gel in Agarose gel electrophoresis. On the

no DNA, there can be no increase in DNA, and no band will contrary, the Sample tube contained a single band. That band is the band we created using PCR. There is only one band because we only synthesized one size of DNA and our

synthesized DNA will have the same shape and sequence as

the original (DNA template). As both the new and old DNA are the same size, when we run the gel they will all run the same distance. Therefore, we only see a single band on the gel.