Basic Rules of Primer Design

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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Basic Rules of Primer Design

    1. Make sure that the primers are not self-complementary or complementary to the other primer in the reaction mixture, as this will encourage the formation of hairpins and primer-dimers (PDs) and will compete with the template for the use of primer and reagents. Both hairpins and PDs can interfere with the PCR method and potentially cause inaccurate quantification and even failed PCRs.
Hairpins consist of internal folds caused by base-pairing between nucleotides and are common secondary structures which may result in failed PCRs. A primer dimer (PD) is a potential by-product in PCR and consists of two primer molecules that have attached or hybridized to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. PDs may interfere with accurate quantification in the PCR method.
    1. If possible, make the 3′ end terminate in C or A, as the 3′ is the end which extends and neither the C nor A nucleotide wobbles (unconventional base pairings in RNA molecule that does not conform to traditional Watson-Crick base pairing). This will increase the specificity. Mispriming can be avoided by making the 3′ end slightly AT rich. Again, there are software tools to help design these, such as AutoDimer, available on the National Institutes of Standards and Technology (NIST) website at: Accessed November 19, 2020.
    2. The concentration of each individual primer should be lower than in singleplex reactions, because the cumulative primer concentration in the reaction can become too high. Concentrations greater than 1 µM can cause primers to anneal along non-target regions which will result in the generation of false product. Insufficient concentrations of either primer could result in little or no amplification. In addition, primer pairs for internal controls should be much less than those for the targets.

Hairpin primers: When two regions of the same strand,
usually complementary in nucleotide, form a double helix
that ends in an unpaired loop.