The Polymerase Chain Reaction, commonly known as PCR, is a laboratory technique that allows scientists to create millions of copies of a specific DNA segment from a very small initial sample. This powerful amplification capability has made PCR an indispensable tool across various scientific fields. It sees widespread use in medical diagnostics, such as identifying infectious agents, in forensic science for analyzing DNA evidence, and in fundamental genetic research.
The Central Player: DNA Polymerase
DNA polymerase is a fundamental enzyme responsible for synthesizing new DNA strands. Within living organisms, it plays an important role in DNA replication, the process by which a cell makes exact copies of its entire genome before dividing. This enzyme works by adding individual building blocks, called nucleotides, to a growing DNA chain, specifically to the 3′ end of an existing strand, building the new strand in the 5′ to 3′ direction.
Unique Demands of PCR and Specialized Polymerases
The PCR process involves repeatedly heating DNA samples to high temperatures, often exceeding 90°C (194°F), to separate the double-stranded DNA into two single strands. This denaturation step is essential for exposing the DNA sequences that need to be copied. Most biological enzymes, including typical DNA polymerases found in human cells, would permanently lose their structure and function under such extreme heat.
This high-temperature requirement necessitated the discovery and use of specialized enzymes known as thermostable DNA polymerases. These enzymes are derived from thermophilic microorganisms that naturally thrive in hot environments, such as hot springs. A prime example is Taq polymerase, isolated from the bacterium Thermus aquaticus.
The heat stability of Taq polymerase is important for the cyclic nature of PCR. Unlike conventional DNA polymerases that would need to be replenished after each heating cycle, Taq polymerase remains active and functional through multiple rounds of amplification. This characteristic simplifies the PCR technique, making it efficient and automatable.
How Enzyme Activity Drives PCR
The activity of DNA polymerase is key to driving the PCR process. After the DNA strands are separated by heat, the temperature is lowered, allowing short synthetic DNA sequences called primers to attach to specific regions on the single-stranded DNA templates. These primers define the starting points for DNA synthesis and flank the target region to be amplified.
The DNA polymerase then begins to synthesize new complementary DNA strands by adding deoxynucleotide triphosphates (dNTPs) one by one. These dNTPs, which are the building blocks of DNA (adenine, guanine, cytosine, and thymine), are incorporated by the enzyme following the base-pairing rules. The polymerase extends the primer, creating a new DNA strand that is an exact copy of the template.
This cycle of denaturation, primer annealing, and DNA synthesis is repeated multiple times, typically 25 to 40 cycles. In each cycle, the newly synthesized DNA strands also serve as templates for subsequent rounds of replication. This leads to an exponential increase in the amount of the target DNA segment, effectively doubling the DNA quantity in each cycle.