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The page below is a sample from the LabCE course Real-Time PCR. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Basic Principles of PCR

A polymerase chain reaction (PCR) consists of three steps: denaturation, annealing (hybridization), and extension. Here is how a basic PCR reaction works:
  1. 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.
  2. Then, to perform PCR, the DNA template that contains the target is added to a tube that contains primers, free nucleotides (adenine, guanine, cysteine, thymine), also known as deoxyribonucleotides (dNTPs), and an enzyme called DNA polymerase. This mixture is placed in a PCR machine. The PCR machine increases and decreases the temperature of the sample in automatic, programmed steps.
  3. 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.
  4. 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). At the end of the first cycle, each double stranded DNA molecule consists of one new and one old DNA strand.
  5. 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.