The polymerase chain reaction is a method to amplify a precisely defined DNA fragment. The following outlines will explain the exact procedure:
This drawing represents a typical DNA double helix. The fragment of interest in-between the red lines should be amplified.
To exactly define the starting and end points of the PCR fragment two short DNA sequences of about 18 to 30 nucleotides in length, so called “primers” are required.
As soon as the primers (illustrated in green and blue, respectively) are synthesized, the PCR reaction can start.
To separate the DNA double helix the hydrogen bonds between the complementary nucleotides have to be broken by heating the reaction mixture to a temperature of 96°C. This first step is called “denaturation”.
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For the second step, called “primer-annealing”, the temperature is lowered to approximately 50-65°C. This temperature allows hybridization of the primers to the right position on the respective DNA single strand. The annealing temperature varies from primer to primer and depends on the primer length and on their nucleotide composition. A cytosine-guanine base pair contains three hydrogen bonds, which makes the binding stronger compared to an adenine-thymine pair with only two hydrogen bonds. As a consequence, the annealing temperature of CG-rich primers is higher than that of AT-rich primers.
The annealing temperature is of crucial importance, since the primers can either bind unspecifically if the temperature is too low, or they do
not bind at all in case of too high temperature.
The following picture shows the binding of the primers to their respective target sequence on the DNA template.
For the next step, the so called “polymerization step”, the temperature is raised to 72°C which is the optimum for the enzyme
Taq-DNA polymerase. This protein uses the bound primers as starting point and the DNA single strands as template for the polymerization of complementary nucleotides.
The Taq polymerase is isolated from the bacterium Thermus aquaticus, usually living in geysers, hot founts in island with a temperature of about 72°C. The
advantage of this polymerase is obvious: being used to high temperatures, it remains functional when heating the reaction mixture repeatedly to 95°C.
In the figure below, the newly synthesized DNA strand is represented in orange.
This step finishes the first cycle with already two copies of the fragment of interest being present. The next steps are nothing but a repetition of this cycle before, starting again with the denaturation step.
Primers bind to the original template strands as well as to the newly synthesized DNA. As a result there are already 4 copies of the sought-after DNA fragment present after the second cycle. 40 PCR cycles lead to a milliard-fold amplification of the starting material.
The PCR reaction itself is carried out in a so-called thermal cycler, a machine that precisely increases or decreases the temperature.
To carry out a successful PCR reaction you need a so-called mastermix containing deoxynucleotidetriphosphates, shortly dNTPs or nucleotides, which are the basic modules of the DNA.
Additionally, it contains buffer substances stabilizing the PCR environment and different ions like Mg2+ necessary as co-factors for the polymerase which is also added to the reaction mixture.
The challenge we were facing in our test system is the fact, that the genetic material of the viruses we wanted to investigate, is single-stranded RNA, not DNA.
Since the DNA polymerase can only amplify DNA we had to rewrite the RNA in so-called complementary DNA, cDNA.
This task is performed by an enzyme called reverse transcriptase, which can use RNA as template for the synthesis of DNA. After reverse transcription into cDNA, the amplification with Taq polymerase can start.
We used a “one-step-kit” of the company Qiagen containing all the components required for reverse transcription as well as PCR. A special program of the thermal cycler allows both reactions to be carried out one after the other in the same tube.