Obtaining genetic markers by using double-stringency PCR with microsatellites and arbitrary primers.

A technique to obtain genetic markers is described. PCR is performed at two different annealing temperatures in a single reaction mixture. During the first cycles, a single microsatellite oligonucleotide is used as primer to amplify DNA between two microsatellite regions. The annealing temperature and consequent stringency is then lowered, and in further cycles a random-amplified polymorphic DNA (RAPD) primer exponentially amplifies parts of the previously amplified fragments, which contain sequences complementary to the RAPD primer. The first round of specific amplification is found to be crucial for obtaining consistent, repeatable results, as compared with the results obtained by RAPD alone. This technique was proven to produce genetically informative markers, as revealed by crosses performed with Drosophila mercatorum strains, and it can be used for genetic mapping or population genetics studies.