Robust amplification and ethidium-visible detection of the fragile X syndrome CGG repeat using Pfu polymerase

We report on a method for ethidium bromide detection of FMR1 normal and premutation-size CGG triplet repeats. A set of PCR conditions has been optimized for the polymerase of the hyperthermophilic bacterium, Pyrococcus furiosus. Using this protocol, normal-size alleles ranging from 5 to 50 repeats, as well as most of premutation-size alleles, varying from > 50 to approximately 200 repeats, could be detected by ethidium bromide staining of agarose gels. Since the protocol requires neither autoradiography nor polyacrylamide gel electrophoresis, it promises to provide a rapid means for the exclusion of fragile X syndrome in males with mental retardation.