Overview of PCR Basic Principles
A polymerase chain reaction (PCR) consists of three steps: denaturation, annealing (hybridization), and extension. Typically, PCR consists of several series of repeated temperature changes (approximately 20-40). These repeated temperature changes are called thermal cycles.
A basic PCR reaction works in the following manner:
- First, two short DNA sequences called primers are designed to bind to the start (3’) and end (5’) of the DNA target. The primer nucleic acid sequences are chosen to flank the target region, so that on amplification the target is increased.
- Then, to perform PCR, the DNA template that contains the target is added to a tube that contains primers, free nucleotides (adenine, guanine, cytosine, thymine), also known as deoxynucleotide triphosphates (dNTPs), and an enzyme called DNA polymerase (Taq polymerase is commonly used). This mixture is placed in a PCR instrument which increases and decreases the temperature of the sample in automatic, programmed steps.
- Initially, the mixture is heated (eg. 95°C) to separate (denaturation step) the double stranded DNA template into single strands. The mixture is then cooled (eg. 60-65°C) so that the primers anneal (hybridization), or bind to the DNA template.
- At this point, the temperature is increased to approximately 72°C to optimize DNA polymerase catalyzing the addition of nucleotides to synthesize new strands of DNA (extension step). At the end of the first cycle, each double stranded DNA molecule consists of one new and one old DNA strand.
- PCR then continues with additional cycles that repeat the aforementioned steps. The newly synthesized DNA segments serve as templates in later cycles, which allow the DNA target to be exponentially amplified millions of times.
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