Detection and genotyping of the GAA trinucleotide repeat length is important for the diagnosis and prognosis of Friedreich's ataxia (FRDA). A two-tier genotyping assay with an improved triple-repeat primed PCR (TR-PCR) for alleles < 200 GAA repeats (± 1-5 repeats), and an agarose gel-based, long-range PCR (LR-PCR) assay to genotype expanded alleles > 200 GAA repeats (± 50 repeats) is described. Of the 1236 DNA samples tested using TR-PCR, 31 were identified to have expanded alleles >200 repeats and were reflexed to the LR-PCR procedure for confirmation and quantification. The TR-PCR assay described here, is a diagnostic genotyping assay which reduces the need for further testing. The LR-PCR component is a confirmatory test for true homozygous and heterozygous samples with normal and expanded alleles as indicated by the TR-PCR assay. The use of this two-tier methodology offers a comprehensive evaluation to detect and genotype the smallest and largest number of GAA repeats, improving the classification of FXN alleles as normal, mutable normal, borderline and expanded alleles.

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