Optimization of PCR amplification of maize microsatellite loci

Maize (Zen mays L,.) microsatellite loci are useful as genetic markers beca use they are numerous, occur in every chromosome, and have a high content o f polymorphism information. Furthermore, they can be amplified by PCR and t he resulting fragments resolved on agarose gels. A major problem, however, is that the primers used in their amplification often have different length and nucleic acid content, requiring optimization of PCR amplification prog rams for each locus. We developed "touchdown" PCR programs successfully use d in the amplification of a set of 125-maize microsatellite loci, chosen as representative of all chromosomes. The programs varied both in relation to the annealing temperature (Tm) and primer concentration. Primers could be divided into four groups. A large group included primers that amplified wel l in a basic previously published amplification program but with different primer concentrations. A second group amplified in alternative programs in which the annealing temperatures were changed so as to include the Tm of ei ther both primers or the highest Tm of the primer pair estimated based on t heir nucleotide composition. A third group included primers that amplified in programs with Tm higher than those estimated, and in a fourth group, wit h just two pairs of primers, the opposite situation prevailed.