[HTML][HTML] A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis

E Lyon, T Laver, P Yu, M Jama, K Young… - The Journal of Molecular …, 2010 - Elsevier
E Lyon, T Laver, P Yu, M Jama, K Young, M Zoccoli, N Marlowe
The Journal of Molecular Diagnostics, 2010Elsevier
Population screening has been proposed for Fragile X syndrome to identify premutation
carrier females and affected newborns. We developed a PCR-based assay capable of
quickly detecting the presence or absence of an expanded FMR1 allele with high sensitivity
and specificity. This assay combines a triplet repeat primed PCR with high-throughput
automated capillary electrophoresis. We evaluated assay performance using archived
samples sent for Fragile X diagnostic testing representing a range of Fragile X CGG-repeat …
Population screening has been proposed for Fragile X syndrome to identify premutation carrier females and affected newborns. We developed a PCR-based assay capable of quickly detecting the presence or absence of an expanded FMR1 allele with high sensitivity and specificity. This assay combines a triplet repeat primed PCR with high-throughput automated capillary electrophoresis. We evaluated assay performance using archived samples sent for Fragile X diagnostic testing representing a range of Fragile X CGG-repeat expansions. Two hundred five previously genotyped samples were tested with the new assay. Data were analyzed for the presence of a trinucleotide “ladder” extending beyond 55 repeats, which was set as a cut-off to identify expanded FMR1 alleles. We identified expanded FMR1 alleles in 132 samples (59 premutation, 71 full mutation, 2 mosaics) and normal FMR1 alleles in 73 samples. We found 100% concordance with previous results from PCR and Southern blot analyses. In addition, we show feasibility of using this assay with DNA extracted from dried-blood spots. Using a single PCR combined with high-throughput fragment analysis on the automated capillary electrophoresis instrument, we developed a rapid and reproducible PCR-based laboratory assay that meets many of the requirements for a first-tier test for population screening.
Elsevier