title: Direct detection of expanded trinucleotide repeats using PCR and DNA hybridization techniques.
abstract: Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.
authors.affiliation: Neurogenetics Section, Hospital for Sick Children, Toronto.
authors.forename: A
authors.initials: A
authors.lastname: Petronis
authors.forename: H H
authors.initials: HH
authors.lastname: Heng
authors.forename: Y
authors.initials: Y
authors.lastname: Tatuch
authors.forename: X M
authors.initials: XM
authors.lastname: Shi
authors.forename: T A
authors.initials: TA
authors.lastname: Klempan
authors.forename: L C
authors.initials: LC
authors.lastname: Tsui
authors.forename: T
authors.initials: T
authors.lastname: Ashizawa
authors.forename: L C
authors.initials: LC
authors.lastname: Surh
authors.forename: J J
authors.initials: JJ
authors.lastname: Holden
authors.forename: J L
authors.initials: JL
authors.lastname: Kennedy
mesh_terms: D015139:Blotting, Southern / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D018911:Trinucleotide Repeats
chemical_list: D004247:DNA