title: Robust amplification and ethidium-visible detection of the fragile X syndrome CGG repeat using Pfu polymerase.
abstract: 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.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Texas 77030.
authors.forename: S S
authors.initials: SS
authors.lastname: Chong
authors.forename: E E
authors.initials: EE
authors.lastname: Eichler
authors.forename: D L
authors.initials: DL
authors.lastname: Nelson
authors.forename: M R
authors.initials: MR
authors.lastname: Hughes
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D003937:Diagnosis, Differential; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics; D018628:Gene Dosage; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D017931:DNA Primers; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D004996:Ethidium