Primer and template sequence secondary structures

Secondary structure is most likely to form by Watson-Crick base-pairing. To reduce self-complementarity, a primer is rejected if it has five contiguous base-pairs with the primer complementary sequence. To prevent primer dimer formation, the 4 residues at the 3' end of a primer should not pair with its complementary sequence. Similarly, complementarity between the forward and reverse primers in a primer pair is also evaluated to prevent detrimental inter-primer dimer formation.

To avoid selecting primers from a template sequence region with potential secondary structure, a primer is rejected if it has 9 or more contiguous base-pairs with the target complementary strand. As one additional check, BLAST similarity search is also carried out to compare the primer and the complementary sequence of the target and the score is required to be less than 18.

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