PriSeT: Efficient De Novo Primer Discovery

Marie Hoffmann; Michael T. Monaghan; Knut Reinert

The second set of constraints, C p , is used when evaluating primer pair candidates. One k-mer will be determined to be the forward primer on the minus strand from 5' to 3' direction, and the second k-mer the reverse primer on the plus strand. As mentioned earlier, denaturation and annealing phase temperatures can be adjusted, but we need to ensure that roughly the same amount of forward and reverse primers are dissociated to produce similar amounts of PCR products by allowing not more than five Kelvin differences in their melting temperatures. GC clamp refers to the amount of CG in the 5'-end of a primer. Especially at the 5'-end where the elongation takes place, a primer should bind stably, but not too strongly. Since C and G bind twice as strong as A or T, ideally one or two nucleotides should be C or G, but the last three should not be exclusively A or T (AT tail). For example, · · · CGATA-3 and · · · CGTCA-3 are not suitable, whereas · · · AGTCA-3 is a suitable primer tail. Cross-annealing refers to the tendency that forward and reverse primer sequences form dimers (see Figure 1 b) and d)). The guidelines for avoidance are analogous to ones for self-annealing. The implementation of the constraint checks is described in more detail in the subsequent sections. Table 2 . Chemical Constraints for k-mers. The first set of constraints Cs applies to k-mers irrespective of their later orientation (forward or reverse). The second set of constraints Cp depends on the final orientation and the k-mer matching partner. Cs is applied in the filter & transform step and Cp checks in the combine step. not more than 4 same nt in a row Dinucleotide Repeats not more than 4 dinucleotides Self-annealing less than four consecutive nts and less than 50 % bond participation