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Primers that are attached to each other at the 3’ end

Prevent primers from binding to DNA template

SELF dimer - hybridization of two same primers or

CROSS dimer - hybridization of two different primers

Can be detected after gel electrophoresis (~30-50 bp)

SELF dimer

CROSS dimer

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Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to  unsuitable product .Thus affect primer template annealing in PCR amplification process .They greatly reduce the availability of primers to complentary the DNA tamplet

i) Hairpins: It is formed by intramolecular interaction within the primer. presence of complementary sequences within its length.Thus reduce the efficiency of the reaction by limiting its availability and ability to bind to the target site. Taq DNA polymerase may extend the 3'end of the primer using the 5' end as the template with hairpin loops having loop structure at 3' end of the primer with the 5' end protruding as a single strand. This leads to stabilization of the hairpins, exhaustion of reactants in the mixture and appearance of non-specific products in the reactionthe and should be avoided.

ii) Self Dimer: A primer self-dimer is formed by intermolecular interactions between the two (same sense) primers, where the primer is homologous to itself. Generally a large amount of primers are used in PCR compared to the amount of target gene. When primers form intermolecular dimers much more readily than hybridizing to target DNA, they reduce the product yield. Optimally a 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated generally.

iii) Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, where they are homologous. Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.

A study was made in designing primer which modifies linear primer to be blunt hairpin primer. For a blunt hairpin primer, the 3’-end sequence is target sequence and a universal sequence is added to the 5’-end of the target sequence for the follow-up library construction. In order to form a blunt hairpin structure, about 13 bp complementary to the 3’-end of the target sequence is added to the 5’-end of the universal sequence, therefore the hairpin primer can anneal by itself to be self loop as molecular beacon and completely lock the 3’-end of primer. The blunt hairpin primer has about 13 bp at 3’-end completely paired with the 5’-end, this structure can block the 3’-end to effectively reduce the non-specific amplifications and primer dimers.  blunt hairpin primers have 20 ~ 25 bp paired with the specific templates. The primer-template paired structure is more stable than the hairpin structure.

Evaluating interactions according to traditional Watson-Crick base pairing rules. The results can be visually inspected or saved to a text file. The information output consists of a visual component along with a score that represents the degree of interaction. Predictions of the transition melting temperature (Tm) and free energy of melting (ΔG) are calculated for each of the potential primer-primer interactions. The formation of a single-stranded loop is unfavorable and is typically associated with a positive free energy of formation. The stability of the duplex stem region must be high enough to overcome the destabilizing loop free energy.  loading the sequences into the program and varying any desired parameters, the user then selects either a primer-dimer or hairpin screen. In Primer3, for hairpin structure, number of comparisons is equal to the number of sequences screened, while for primer-dimer, the value is equal to (n2 + n)/2, where n is the total number of primers.

Vallone, P.M. and Butler, J.M., 2004. AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques, 37(2), pp.226-231.

Gräntzdörffer A., Nemetz C. (2003) Reduction of Primer-Dimer Formation during Generation of Expression Fragments by PCR. In: Swartz J.R. (eds) Cell-Free Protein Expression. Springer, Berlin, Heidelberg