Self-complementarity can lead to stable hairpin formation with just four GC base pairs in the stem and three bases in the loop. If oligonucleotides form hairpins (intramolecular hybridization), they are not available for hybridization to the target regions. Any kind of hairpin structure should be avoided in a primer.
Click here to view an animation on hairpins.
The available nucleotides can be thought of as forming a smaller, and therefore less specific, primer because the entire primer is not used to discriminate among target sequences. If the 3′ nucleotides bind strongly, any template sequences that are complementary to the 3′ end are amplified. Conversely, if binding is strongest at the 5′ end, the typing binding event on the template DNA begins at the 5′ end. Polymerases, however, cannot begin elongation until the 3′ end binds. Therefore, the entire primer is used to distinguish among target sequences.
AutoDimer is a program that was originally created to assist in the development of multiplex PCR assays for probing STR and SNP markers for forensic science purposes. The program rapidly screens previously selected PCR primers for primer-dimer and hairpin interactions in short DNA oligomers (< 30 nucleotides).13
Click here to visit the AutoDimer homepage at the National Institute of Science and Technology.
In extreme cases of complementarity, a gapped duplex can form when the primer and target are completely complementary except for a few bases.
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