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 an d premutation-size CGG triplet repeats. A set of PCR conditions has be en optimized for the polymerase of the hyperthermophilic bacterium, Py rococcus furiosus. Using this protocol, normal-size alleles ranging fr om 5 to 50 repeats, as well as most of premutation-size alleles, varyi ng from >50 to approximately 200 repeats, could be detected by ethidiu m bromide staining of agarose gels. Since the protocol requires neithe r autoradiography nor polyacrylamide gel electrophoresis, it promises to provide a rapid means for the exclusion of fragile X syndrome in ma les with mental retardation. (C) 1994 Wiley-Liss, Inc.