title: A novel PCR-based approach for the detection of the Huntington disease associated trinucleotide repeat expansion.
abstract: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder associated with expansions of an unstable CAG trinucleotide repeat in exon 1 of the IT15 gene. In normal individuals, IT15 contains up to 35 CAG repeats, while in affected the repeat length is >36. Polymerase chain reaction (PCR) is used to estimate the number of CAG repeats but may be inefficient in long repeats because of the high C+G content of the HD locus. We present a novel PCR approach for the diagnosis of HD, which permits direct visualization of the amplified products on agarose gel, using ethidium bromide. It is based on the methylation-sensitive conversion of C residues to U by bisulfite treatment of single-stranded DNA and subsequent amplification of the sense strand with specific primers. The bisulfite treatment dramatically reduces the C + G content of the region; thus, the high Tm and stable secondary structures are no longer obstacles to PCR. In both normal and affected individuals, UAG repeats (5'- CAG-3', before bisulfite treatment) in the sense strand can easily be amplified and visualized on a gel by ethidium bromide staining. The method has considerable advantages compared with other described PCR-based diagnostic tests for HD.
authors: Department of Clinical Genetics, University Hospital, Lund, Sweden. ioannis.panagopoulos@molmed.lu.se
authors: I
authors: I
authors: Panagopoulos
authors: C
authors: C
authors: Lassen
authors: U
authors: U
authors: Kristoffersson
authors: P
authors: P
authors: Aman
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D006816:Huntington Disease / Q000175:diagnosis / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013447:Sulfites / Q000378:metabolism; D019680:Trinucleotide Repeat Expansion
chemical_list: D013447:Sulfites; C042345:hydrogen sulfite