Properties of sequence-tagged-site primer sets influencing repeatability

The polymerase chain reaction (PCR) has become a standard procedure in plan t genetics, and is the basis for many emerging genomics approaches to mappi ng and gene identification. One advantage of PCR is that sequence informati on for primer sets can be exchanged between laboratories, obviating the nee d for exchange and maintenance of biological materials. Repeatability of pr imer sets, whereby the same products are amplified in different laboratorie s using the same primer set, is important to successful exchange and utiliz ation. We have developed several hundred sequence-tagged site (STS) primer sets for wheat and barley. The ability of the primer sets to generate repro ducible amplifications in other laboratories has been variable. We wished t o empirically determine the properties of the primer sets that most influen ced repeatability. A total of 96 primer sets were tested with four genomic DNA samples on each of four thermocyclers. All major bands were repeatable across all four thermocyclers for approximately 50% of the primer sets. Cha racteristics most often associated with differences in repeatability includ ed primer GC content and 3prime-end stability of the primers. The propensit y for primer-dimer formation was not a factor in repeatability. Our results provide empirical direction for the development of repeatable primer sets.