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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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Primers

Primers, are short synthetic DNA fragments that are complementary to the sequences flanking either side of the desired target region to be copied. In general, primers are 18–25 base pairs in length are used to provide a practical size.
Primer pairs should have compatible melting temperatures (within 5°C) and contain approximately 50% GC content. Primers with high GC content can form stable imperfect hybrids. Conversely, high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3' end of the primer should be GC rich (GC clamp) to enhance annealing of the end that will be extended. It is critical to analyze primer pair sequences to avoid complementarity and hybridization between primers (primer-dimers).
Primers can either be purchased or produced in the laboratory. See image for a graphic representation.