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What secondary structure considerations need to be included when designing primers for PCR?

When designing primers for PCR, two types of secondary structures should be analyzed: dimers and hairpins. For dimers, both self- and hetero-dimers should be reviewed. In general, the ΔG value for dimer analysis should be between 0 to −9 kcal/mole for optimal design. Values more negative than this may adversely affect PCR reactions. For hairpins, the Tm of the hairpin should be lower than the annealing temperature for the reaction. The Tm for the strongest hairpin should be at least 50°C below the annealing temperature. These secondary structures and their corresponding stabilities can be calculated online with the IDT SciTools® OligoAnalyzer.

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OligoAnalyzer