Among the millions of DNA fragments that result are some containing the section of the FMR1 gene with the CGG repeat. When there is no mutation, these FMR1 fragments are of a single known size. If a mutation is present, the fragments are longer. Furthermore, fragments from FMR1 genes that are methylated can be distinguished by size from those that are unmethylated because one of the enzymes utilized fails to cut if methylation is present. Each patient DNA sample is applied to a slot at the top of the gel and runs down a narrow strip of the gel.
Short fragments move faster and further than long fragments.
The resulting long smear of DNA fragments can be visualized by staining with a dye that glows bright orange. At this point the FMR1 fragments are indistinguishable from all the other DNA fragments. The smears of DNA fragments are transferred onto a thin sheet of nylon by blotting and then the sheet is bathed with a radioactively-labeled FMR1 gene probe. The probe singles out the matching FMR1 gene fragments and binds tightly. Exposure of the nylon sheet to X-ray film results in short black horizontal lines on the film corresponding to the positions of the patient's FMR1 gene fragments.