Trinucleotide Repeat Protocols

Volume 277 of the series Methods in Molecular Biology™ pp 61-76

Analysis of Unstable Triplet Repeats Using Small-Pool Polymerase Chain Reaction

  • Mário Gomes-PereiraAffiliated withDivision of Molecular Genetics, Institute of Biomedical and Life Sciences, Anderson College Complex, University of Glasgow
  • , Sanjay I. BidichandaniAffiliated withDepartments of Biochemistry & Molecular Biology and Pediatrics, University of Oklahoma Health Sciences Center
  • , Darren G. MoncktonAffiliated withDivision of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College Complex

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Small-pool polymerase chain reaction (PCR) constitutes the PCR amplification of a trinucleotide repeat in multiple small pools of input DNA containing in the order of from 0.5 to 200 genome equivalents. Products are resolved by agarose gel electrophoresis and detected by Southern blot hybridization under conditions that allow the identification of products derived from single-input molecules. The method allows the detailed quantification of the degree of repeat-length variation in a given sample, including the detection of common variants and those alleles present only in a small subset of cells. Detailed analysis of repeat dynamics is essential for a complete understanding of the molecular mechanisms that generate diversity and lead to disease in the unstable trinucleotide DNA repeat disorders.

Key Words

Mutation polymerase chain reaction trinucleotide repeat myotonic dystrophy Huntington’s disease Friedreich ataxia spinocerebellar ataxia small-pool PCR unstable DNA expansion repeat dynamics Southern blot triplet repeat sperm microsatellite