The polymerase chain reaction (PCR) copies DNA, utilizing repeated cycles of three basic steps. This reaction utilizes Taq DNA polymerase enzyme, which is a recombinant, thermostable DNA polymerase from the organism Thermus aquaticus.
Step 1: Denaturation
After an extraction process designed to release DNA from cellular material, an aliquot of the extracted sample is added to a reaction mixture which contains polymerase enzyme, forward and reverse primers for the target of interest, and nucleotides. During the first step, this mixture is heated (generally to 95°C). This causes complementary strands of DNA to separate (denaturation).
Step 2: Annealing
The reaction mixture is cooled to 55°C. During this annealing phase, if the target of interest is present in the patient sample, the primers will bind to their complementary sequences of DNA. Primers are short sequences of single stranded DNA that mark both ends of the target sequence. Two primers are utilized, one for each of the complementary single strands of DNA released during denaturation. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand). The 5' ends of both primers bind to the 3' end of each DNA strand.
Step 3: Synthesis at 72°C
The temperature is raised, typically to a temperature of 72°C. At this temperature the polymerase enzyme begins the process of DNA synthesis. Free nucleotides, complementary to the bases in each strand, are added sequentially to both the sense and anti-sense strands. Synthesis always occurs from the 5' to the 3' direction on each primer. This results in the simultaneous synthesis of two new strands of DNA: in the direction from the start codon to the stop codon from the forward primer, and in the direction from the stop codon to the start codon from the reverse primer.
These steps are repeated in cycles, resulting in a geometric doubling of the target sequence at the end of each cycle. A typical assay will utilize around 45 cycles. Once amplification is completed, detection and identification of the multiplied target occurs.
