Arsh Chopra

The Molecular Theory behind the Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction (PCR) is an automated method of producing millions of copies of a specified DNA fragment from a single or few original copies. The technique is commonly employed in research laboratories within the governmental, private, and academic sectors. PCR can be easily employed by any individual with a basic background in molecular biology. The machines (see Figure 1) are used by people with a range of experience, from undergraduate students to full time researchers. PCR works on the basis of DNA complementarity: the idea that a given nucleotide (the functional unit of DNA) will only bond with one specific other nucleotide. The principle steps of PCR include (1) denaturation of template DNA, (2) annealing of primers to template DNA strands, and (3) extension (synthesis) of new DNA strands.

Figure 1. Ampicon ThermoMix 500 PCR Machine Courtesy of: http://www.wisbiomed.com/ec/images/mix500-new.jpg

Denaturing the Template DNA The PCR reaction begins with denaturing the template DNA. In nature, DNA occurs as a double stranded molecule, and must be separated before it can be copied. The structure of a typical DNA molecule can be seen in Figure 2 (right). During the denaturing phase, the DNA samples are heated to 94-96 C for several minutes. In doing so, the hydrogen bonds connecting the two strands at every base pair are broken, and the DNA separates into two single strands. Because the individual strands are bound together prior to separation, the individual bases cannot be recognized and subsequently copied. Therefore, this denaturing step is critical for replication. Primer Annealing Upon denaturing the DNA, the nucleotide bases of the template strand are exposed to the reaction environment. As a component of the reaction cocktail, short fragments of DNA that have been commercially synthesized are present. These fragments, known as primers, can be synthesized with any sequence (nucleotide pattern) according to the needs of the researcher. As such, these primers will only match a specific segment of the template DNA. Primers complementary to the beginning and end of the desired fragment are used, to ensure that only that region in between the primers is replicated. By lowering the temperature of the samples to
Figure 2. Typical DNA Structure Courtesy of: http://www.biologycorner.com/resource s/DNA-colored.gif

45-60 C for several minutes, the primers will readily anneal (bond) to the exposed template strand (Figure 3).

Figure 3. Annealing of primers to template DNA strands. Courtesy of: http://www.cryst.bbk.ac.uk/pps97/assignments/projects/borek/Domina/anneal2.gif

Primer Extension and Synthesis The final step in PCR is the extension of the primers into newly synthesized DNA strands. During this final stage, the temperature of the samples is raised to 72 C for two minutes, the optimal environment for synthesis. Free floating nucleotides (dNTPs) present in the reaction mixture are added to the existing primers by an enzyme called Taq Polymerase. An illustration of this process can be seen in Figure 4, below.

Figure 4. Extension of primers on new DNA strand. Courtesy of: http://users.ugent.be/~avierstr/principles/pcrsteps.gif

Upon completion of this step, a single DNA fragment consisting of two individual strands will be

successfully replicated into two new fragments. Each original strand will be preserved and have a newly synthesized strand complementary to it. However, this represents only a single cycle of the Polymerase Chain Reaction. This cycle is repeated 30-40 times, and will result in an exponential increase in the number of identical DNA fragments (Figure 5). Whereas the first cycle copies a single fragment into two, the second cycle will copy
Figure 5. Exponential growth of DNA copies Courtesy of: http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne000 0/15828879/f1_allanmax.jpg

two into four, the third cycle will copy four into eight, and so on. As a result, the number of copies of DNA rapidly increases from the original template. Conclusion Through a series of temperature changes, DNA can be modified and artificially replicated using the Polymerase Chain Reaction. The initial denaturing allows for modifications to be made to the DNA of interest, and is followed by attachment of primers to specify the exact location of amplification. Finally, the addition of new nucleotides by Taq Polymerase completes the synthesis of a new DNA strand with a sequence complementary to the template. These steps are repeated multiple times to produce millions of copies of the DNA molecules, allowing for expanded research in molecular studies.