Target-Specific Primer Design

As an example for target-specific design, sequences of Ac transposons from Z. mays were downloaded from GenBank. Sequences of individual Ac transposons were extracted and used to build multiple alignments. Regions within both termini that were highly conserved in all Ac transposons were used to target the SSPs. Sets of Ac-specific primers were designed to target the termini of the selected transposon. The SSPs are shown in Table 3. The following rules were used for selection of the SSPs: each SSP should be 25–35 nt, have a GC content of 40–60%, and a calculated T_m ≥ 65°C. A good SSP should not be capable of forming self-dimers. Generally, the Ac-specific primers should be as close to the end of the known sequence as possible. The SSPs were designed using FastPCR software with the following calculation parameters: 50 mM monovalent cation (K⁺/NH₄⁺), no divalent cations, and a working primer concentration of 0.25 μM.

Sequence-specific primers (SSPs) for the Ac transposon from Zea mays.