A novel PCR-based approach for the detection of the Huntington disease associated trinucleotide repeat expansion

Hum Mutat. 1999;13(3):232-6. doi: 10.1002/(SICI)1098-1004(1999)13:3<232::AID-HUMU7>3.0.CO;2-N.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Mutational Analysis / methods*
  • Electrophoresis, Agar Gel
  • Humans
  • Huntington Disease / diagnosis
  • Huntington Disease / genetics*
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Sulfites / metabolism
  • Trinucleotide Repeat Expansion*

Substances

  • Sulfites
  • hydrogen sulfite