A revolutionary method in amplifying many copies of a specific small sequence of DNA from a longer DNA molecule has greatly influenced the molecular biology world. By 1980, many factors that were needed for the polymerase chain reaction were already discovered, but it was not until in 1983 that Kary B. Mullis perceived the idea of this breakthrough technique. With knowledge about primers annealing to a complementary sequence of DNA, Mullis’ initial experiments of PCR involved him having to self-adjust the temperatures during the processes of DNA denaturation, annealing, and elongation, whilst also adding in new DNA polymerases for every cycle. This proved to be very tedious and time consuming, until the purification of Taq polymerase in 1985. This heat stable DNA polymerase was able to sustain the high temperatures of denaturation, and could efficiently elongate DNA strands through numerous cycles. Then, in 1987, the first PCR thermal cycler machine allowed for the regulation of temperature and timing, which significantly reduced the costs and hours of manually adjusting the different processes of PCR that Mullis originally had endured.
In order to perform PCR, several ingredients need to be included, which are: a template DNA strand, forward and reverse primers, dNTPs, a thermostable DNA polymerase enzyme, a reaction buffer, MgCl2, water, and a thermal cycler. The template DNA strand contains the target DNA sequence that is going to be amplified. The forward and reverse primers are synthetic DNA molecules that have sequences that will anneal complementary to the template DNA strand, binding to the two ends that surround the DNA sequence of interest. For PCR, only a specific region is going to be amplified, which will be from the 5′ end of the forward primer, to the 5′ end of the reverse primer, thus it is vital to have well designed primers. When designing primers, a few factors come into play, in order for both forward and reverse primers to function effectively during PCR. They must be complementary to the template DNA, their length are designed to be between 20 and 30 nucleotides long, which allows them to be sufficiently specific to the region of DNA. In addition, both primers should have similar melting point temperatures, which can be calculated based on their length and number of A and T nucleotides relative to the number of C and G nucleotides in the sequence. A thermostable DNA polymerase enzyme, such as Taq polymerase, will function at high temperatures, and not degrade during DNA denaturation; it will read the DNA template and assemble the dNTPs, which are the nucleotides used to make up the new DNA. A reaction buffer, such as TBE buffer, will allow for maintaining an ideal pH balance, while MgCl2 will reduce charge repulsion between the primer and the template strand and is a required cofactor for the Taq polymerase; adding too little cation may result in difficult annealing and reduce the amount of product, while adding too much cation may reduce specificity of annealing and produce non-specific …
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