Basic Rules of Primer Design - LabCE.com, Laboratory Continuing Education

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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

Make sure that the primers are not self-complementary or complementary to the other primers in the reaction mixture. This will encourage the formation of hairpins and primer-dimers (PDs) and will compete with the template for using 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 that may result in failed PCRs. A primer dimer (PD) is a potential by-product in PCR. It consists of two primer molecules 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 the amplification of the DNA sequence targeted for PCR amplification. PDs may interfere with accurate quantification in the PCR method.

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 do not conform to traditional Watson-Crick base pairing). This will increase the specificity. Mispriming can be avoided by making the 3′ ends 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: https://strbase-archive.nist.gov/AutoDimerHomepage/AutoDimerProgramHomepage.htm
The concentration of each 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, resulting in false product generation. 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.