METHODS AND TECHNOLOGIES

This specific method is used to determine the number of a variable sequence motif repetitions such as “CCG,” which can occur with different allele lengths (e.g., paternal allele: 19 x CCG, maternal allele: 20 x CCG).

Fluorescently-labeled PCR products, prepared using a fluorescence-labeled primer (e.g., blue fluorescence) and a fluorescence-labeled internal standard (e.g., red fluorescence), are separated by size in a polymer-filled capillary using capillary gel electrophoresis. As the products pass through the detection unit, a laser excites the fluorescence, and the resulting emissions are detected. The internal standard facilitates the subsequent determination of fragment lengths using specialized software.

This technique is used in the molecular genetic analysis of conditions like triplet repeat disorders, including fragile X syndrome and spinocerebellar ataxias (types 1, 2, 3, 6, 7, and 17). It helps determine whether a patient with relevant clinical symptoms has a pathogenic triplet repeat expansion.

Recognized as a well-established, reliable, and robust tool in genetic diagnostics, this method has been in use for some time. The triplet repeat count is typically reported within a margin of error specific to the sequence being analyzed (e.g., for normal alleles in the ATXN1 gene: ±1 (29±1 CAG repeats)). Only limited interpretation is possible for alleles that appear to be homozygous. In such scenarios, a triplet repeat-primed PCR (TP-PCR) should be conducted to rule out the presence of a second, highly elongated allele that may not have been amplified in the standard reaction.