Knowledge of the CGG•CCG-repeat number, AGG interruption status, and the extent of DNA methylation of the FMR1 gene are vital for both diagnosis of the fragile X-related disorders and for basic research into disease mechanisms. We describe here assays that we use in our laboratory to assess these parameters. Our assays are PCR-based and include one for repeat size that can also be used to assess the extent of methylation and a related assay that allows the AGG interruption pattern to be reliably determined even in women. A second more quantitative methylation assay is also described. We also describe our method for cloning of repeats to generate the reference standards necessary for the accurate determination of repeat number and AGG interruption status.
Keywords: AGG PCR assay; AGG interruptions; FMR1; FMR1-related disorders (FMR1 disorders); FX-associated primary ovarian insufficiency (FXPOI); FX-associated tremor and ataxia syndrome (FXTAS); Fragile X syndrome (FXS); Quantitative methylation assay; Repeat PCR; qPCR.