Triplet primed repeat PCR a?? An approachable diagnosis method for detecting triplet repeat disorders -Friedreich Ataxia

摘要

Diagnostic tests have been developed to detect triplet repeat expansion associated with many neurodegenerative triplet repeat disorders employing techniques including Southern blotting and PCR amplification of the gene expansion. Lack of precision and accuracy in determining the exact size of GAA repeats (triplet repeats in case of Friedreich ataxia) by conventional PCR amplification procedure led to improvisation of this method. This study was aimed to standardize a diagnostic procedure for detecting the GAA repeat expansion in suspected patients of Friedreich ataxia using improvised triplet Primed Repeat PCR technique. DNA samples required for the study was isolated from white blood cells of 50 healthy subjects and 5 suspected cases of Friedreich Ataxia, the isolated DNA were quantified and was used for performing the triplet primed PCR . The PCR products were screened through Capillary Electrophoresis to detect the exact number of GAA repeats present in the DNA samples. Among triplet repeat primed PCR performed in 50 control samples and 5 suspected FRDA patients, all healthy control subjects analyzed showed the electropherogram with peaks in normal limits and the suspected cases showed the peaks above normal repeat levels suggesting the pathogenic expansion. These findings suggests that the triplet repeat primed PCR assay can be used as a reliable and faster diagnosis technique to detect the pathogenic repeats expansions observed in triplet repeat disorders.