Primer or target template secondary structures can retard primer annealing, leading to decreased amplification efficiency. The likelihood of secondary structure is greatest in regions rich in complementary base pairing, such as the stem of a stem-loop structure (Mount, 2001). If part of a primer or target sequence is inaccessible due to secondary-structure formation, the primer annealing efficiency may decrease dramatically.
In addition, primer secondary-structure may lead to primer dimer formation, which is one of the biggest challenges for accurate quantification by real-time PCR, especially when DNA intercalating dyes (e.g., SYBR® Green I) are used. Primer dimers can be produced by primer self-annealing, or by annealing between the forward and reverse primers. Therefore, the forward and reverse primers should be evaluated together during design to avoid potential primer dimer formation.
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